Chapter 1 Introduction
CLONING, EXPRESSION AND PURIFICATION OF Toxocara canis RECOMBINANT ANTIGENS (rTES-26, rTES-32, rTES-120) AND DEVELOPMENT OF SERODIAGNOSTIC TEST FOR TOXOCARIASIS
SUHARNI BINTI MOHAMAD
UNIVERSITI SAINS MALAYSIA 2009
1 CLONING, EXPRESSION AND PURIFICATION OF Toxocara canis RECOMBINANT ANTIGENS (rTES-26, rTES-32, rTES-120) AND DEVELOPMENT OF SERODIAGNOSTIC TEST FOR TOXOCARIASIS
by
SUHARNI BINTI MOHAMAD
Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy
April 2009
2 DEDICATIONS
To
My husband, Mohd Rozi Aziz,
My mother, Nik Hanizan and my mother-in-law, Zainab,
My father, Mohamad and my father-in-law, Aziz
My sons, Mohamad Rasydan Hakimi, Mohamad Rafsyan Hakim, Mohamad Rahaizat
Hakimin and Mohamad Raqwan Hatim
3 ACKNOWLEDGEMENTS
All praises and gratitude are due to Allah, the Most Merciful and Compassionate.
First and foremost, I wish to express my heartfelt gratitude to my supervisor,
Professor Rahmah Noordin, who is generous in sharing her knowledge and in giving advice.
I sincerely thank her for her steadfast encouragement, constructive comments, suggestions, criticism during the writing of this thesis and for guiding me to be a good scientist.
My sincere appreciation to Profesor Asma Ismail, lecturers and staff of INFORMM,
Penang and Kelantan for their support, invaluable suggestions and technical assistance throughout my study. I am deeply grateful to INFORMM for providing well-equipped infrastructure and facilities to carry out the project.
I also would like to acknowledge the Director of Veterinary Laboratory in Kota
Bharu, Dr. Naheed; Director of Veterinary Research Institute in Perlis, Dr Tareq; Dr
Norhana from Veterinary Laboratory in Bukit Tengah, Penang; Seberang Prai City Council,
Penang; and staff of Veterinary Laboratories at Bukit Tengah, Penang, Kota Bharu, Kelantan and Kangar, Perlis for their technical assistance and for providing the puppies and stray dogs from which adult T. canis worms were collected that made this study possible. My sincere thanks to the late Associate Prof. Dr. Afifi Sheikh Abu Bakar for his assistance in the identification of T. canis worms, Dr. Lim Boon Huat from School of Health Sciences and Dr
Azlina from International Medical University for helping in statistical analysis. I would also like to thank the Department of Microbiology and Parasitology, School of Medical Sciences,
USM; Department of Parasitology, Faculty of Medicine, University of Malaya, Health
Centre at Universiti Sains Malaysia (USM), Penang and the Scottish Parasite Diagnostic
Laboratory (SPDL) for providing the serum samples.
4 I would like to extend my warm and sincere thanks to my friends and colleagues, Mr
Mehdi, K.Ana, K.Syikin, Emelia, Ude, Cheah, Syida, Zul, Nyambar, Nurul, Madihah,
Anizah for their friendship, help, support, technical assistance, encouragement and advice.
On a personal note, I am forever indebted to my family for their never-ending love, care, support, encouragement and patience. This thesis is specially dedicated to my beloved mum who had given me her undivided attention in all my undertakings, to my husband and all my children who had made my life wonderful, Mohamad Rasydan Hakimi, Mohamad
Rafsyan Hakim, Mohamad Rahaizat Hakimin and Mohamad Raqwan Hatim, and to my siblings, Suhaili, Suhaiza, Suhaila and Nor Azrani, who have been a great source of inspiration.
This project was funded by the IRPA research grant under Profesor Rahmah
Noordin, EA No 06-02-05-4261 EA019. I would also like to thank the National Science
Fellowship from the Malaysian Ministry of Science, Technology and Innovation (MOSTI) for the financial support during my study.
5 TABLE OF CONTENTS
Page no.
DEDICATIONS…………………………………………………………………….……….ii
ACKNOWLEDGEMENTS…………………………………………………..……………iii
TABLE OF CONTENTS……………………………………………………..…………….v
LIST OF TABLES…………………………………………………………………...... xi
LIST OF FIGURES……………………………………………………………………....xiii
LIST OF ABBREVIATIONS & ACRONYMS ..……………………………….………xvi
ABSTRAK………………………………………………………………………….……..viii
ABSTRACT……………………………………………………………………………….xxi
CHAPTER 1 INTRODUCTION
1.1 General introduction……………………………………………………………..….1 1.2 The taxonomy of Toxocara and morphology……………………………………….2 1.3 The larval surface coat…………………………………………………………...... 3 1.4 Toxocara excretory/secretory antigens (TES)………………………………………6 1.5 Life cycle of T. canis…………………………………………………………....…..7 1.6 Egg survival…………………………………………………………………....…...11 1.7 Global seroprevalence of human toxocariasis……………………………………...12 1.8 Signs and symptoms of toxocariasis……………………………………………….14 1.8.1 Visceral larva migrans (VLM)...... 15 1.8.2 Ocular larva migrans (OLM)………………………………………………16 1.8.3 Covert toxocariasis (CT)………………………………………………...... 17 1.8.4 Neurological toxocariasis…………………………………………...... 18 1.9 Pathogenesis of human toxocariasis………………………………………………..18 1.10 Immunological aspect of toxocariasis……………………………………………...20 1.10.1 Cellular immune responses………………………………………………..20 1.10.2 Antibody subclasses in toxocariasis………………………………….……20 1.10.2.1 IgM………………………………………………...……………20 1.10.2.2 IgE………………………………………………………………21 1.10.2.3 IgG subclasses…………………………………………..………22
6 1.11 Diagnosis of human toxocariasis…………………………………………...... 24 1.11.1 Clinical diagnosis…………………………………………………...... 24 1.11.1.1 Radiology……………………………………………….………24 1.11.2 Laboratory diagnosis………………………………………………………25 1.11.2.1 Parasitology tests…………………………………………...... 25 1.11.2.2 Serology tests…………………………………………………...25 1.11.2.2.1 IgG antibody avidity…………………..………...28 1.11.2.2.2 Western blot (WB)………………………………29 1.11.2.3 Rapid test…………………………………………………….…30 1.11.2.4 Other assays…………………………………….………………31 1.11.3 Diagnosis of OLM…………………………………………………………32
1.11.3.1 Clinical diagnosis………………………………………………..32 1.11.3.2 Laboratory diagnosis……………………………………...... 32 1.12 Recombinant TES antigens in serodiagnosis…..……………………………………34 1.12.1 rTES-30…………………………………………………………………...34 1.12.2 rTES-120……………………………………………………………….....35 1.13 Treatment, management and prognosis……………………………………………...36 1.14 Statement of the problem and rationale of the study………………………………...36 1.15 Objectives of the study………………………………………………………………39
CHAPTER 2 GENERAL MATERIALS AND METHODS
2.1 Materials……………………………………………………………………………..41 2.1.1 T. canis worms…………………………………………………………..….41 2.1.2 Oligonucleotide primers………………………………………..………..….41 2.1.3 Bacterial strains………………………………………………...…….……..42 2.1.4 Cloning and expression vectors…………………………………...... 42 2.1.5 Human serum samples……………………………………………………....42 2.1.6 Chemicals, reagents and media………………………………………….…..44 2.1.7 Sterilisation……………………………………………………………….…44 2.1.7.1 Moist heat……………………………………………………… .. ..44 2.1.7.2 Membrane filtration………………………………………………...44 2.1.7.3 DEPC treatment…………………………………………………….44
2.1.8 Preparation of common media………………………………………………45 2.1.8.1 Luria-Bertani (LB) broth……………………………………………45 2.1.8.2 Terrific broth (TB)………………………………………………….45 2.1.8.3 Salt solution………………………………………………………...45 2.1.8.4 LB agar……………………………………………………………..45 2.1.8.5 LB agar with ampicillin…………………………………………….46 2.1.8.6 LB agar with kanamycin…………………………………………....46
2.1.9 Preparation of common buffers and reagents……………………………….46 2.1.9.1 Phosphate buffered saline (PBS), pH 7.2…………………………46 2.1.9.2 PBS-Tween 20 (PBS-T), 0.05% (v/v)…………………………….46 2.1.9.3 Tris buffered saline (TBS)………………………………………...46 2.1.9.4 TBS-Tween 20 (TBS-T), 0.05% (v/v)…………………………….47
7 2.1.9.5 TBS/Tween-20/Triton-X (TBSTT) (w/v/v)…………………….....47 2.1.9.6 NaOH solution (3M)………………………………………………47 2.1.9.7 HCl solution (1M)…………………………………………………47 2.1.9.8 EDTA solution, 0.5 M (pH 8.0)…………………………………...47 2.1.9.9 Ampicillin stock solution (100 mg/ml)……………………………47 2.1.9.10 Kanamycin stock solution (30 mg/ml)……………………………48 2.1.9.11 Magnesium chloride (MgCl2), 100 mM...... 48 2.1.9.12 Calcium chloride (CaCl2), 100 mM...... 48 2.1.9.13 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 20 mg/ml (w/v)…………………………………………...... 48 2.1.9.14 Isopropyl-β-D- galactopyranoside (IPTG), 800 mM …...... 48 2.1.9.15 Ethanol (70%)……………………………………………………..48
2.1.10 Preparation of reagents for culture of L2 larvae……………………………49 2.1.10.1 Normal saline [sodium chloride, 0.85% (w/v)] …………………...49 2.1.10.2 RPMI-1640 stock solution………………………………………..49 2.1.10.3 Acid-pepsin solution [Pepsin 1% (w/v), HCl 0.1% (v/v)]……….49 2.1.10.4 DEPC-treated water………………………………………………49
2.1.11 Preparation of reagents for agarose gel electrophoresis……………………...50 2.1.11.1 Tris-Borate-EDTA (TBE) buffer (5X)…………………………...50 2.1.11.2 Ethidium bromide solution (EtBr), 10 mg/ml (w/v)……………..50 2.1.11.3 Agarose gel loading dye…………………………………………50
2.1.12 Preparation of reagents for SDS-PAGE……………………………………50 2.1.12.1 Resolving gel buffer, pH 9.3…………………………………….50 2.1.12.2 Stacking gel buffer, pH 6.8………………………………………51 2.1.12.3 2X sample buffer…………………………………………………51 2.1.12.4 Ammonium persulfate (AP), 20% (w/v)…………………………51 2.1.12.5 Running buffer…………………………………………………...51 2.1.12.6 Coomassie blue stain…………………………………………….52 2.1.12.7 Coomassie destaining solution…………………………………..52
2.1.13 Preparation of reagents for immunodetection………………………………52 2.1.13.1 Western blot transfer buffer…………………………………… ..52 2.1.13.2 Ponceau S stain…………………………………………………...52 2.1.13.3 Amido black stain………………………………………………...52 2.1.13.4 Blocking stock solution, 10% (w/v)……………………………...53 2.1.13.5 Chemiluminescence substrate …………………………………….53
2.1.14 Preparation of reagents for histidine-tagged protein purification under native condition………………………………………………..……………53 2.1.14.1 Protease inhibitor cocktail, 0.37 mg/ml……………………….…53 2.1.14.2 Lysozyme stock (10 mg/ml) (w/v)…………………………….....53 2.1.14.3 DNAse 1 (w/v)…………………………………………………...54 2.1.14.4 10 mM lysis buffer (containing 300 mM NaCl)……………….....54 2.1.14.5 20 mM wash buffer (containing 300 mM NaCl)…………………54 2.1.14.6 30 mM wash buffer (containing 300 mM NaCl)…………………54 2.1.14.7 40 mM wash buffer (containing 300 mM NaCl)………………....54 2.1.14.8 Elution buffer (containing 300 mM NaCl)…………………..…...55 2.1.14.9 10 mM lysis buffer (containing 500 mM NaCl)………………….55 2.1.14.10 20 mM wash buffer (containing 500 mM NaCl)………………....55 2.1.14.11 30 mM wash buffer (containing 500 mM NaCl)……………..…..55
8 2.1.14.12 40 mM wash buffer (containing 500 mM NaCl)…………………56 2.1.14.13 Elution buffer (containing 500 mM NaCl)……………………....56 2.1.14.14 Stripping buffer……………………………….………………..…56
2.1.15 Preparation of reagents for GST-tagged protein purification under native condition……………………………………………………………...56 2.1.15.1 GST bind/wash buffer (1X)...... 56 2.1.15.2 GST elution buffer (10X) (w/v)……………………………….…57
2.1.16 Preparation of reagents for ELISA………………………………………….57 2.1.16.1 Coating buffer……………………………………………………57 2.1.16.2 Blocking stock solution, 10%...... 57 2.1.16.3 Substrate 2,2’-Azino-d-[3-ethylbenthiazoline sulfonate] (ABTS)...... 57
2.2 Methods……………………………………………………………………………… 58 2.2.1 Isolation and cultivation of T. canis ova………………………...... 58 2.2.1.1 Storage of T. canis larvae…………………………………………..59 2.2.2 Plasmid extraction………………………………………………...... 61 2.2.3 Quantification of nucleic acids……………………………………………....62 2.2.4 Polymerase chain reaction (PCR)…………………………………………....62 2.2.5 DNA agarose gel electrophoresis……………………………………………63 2.2.6 Purification of PCR products………………………………………………..63 2.2.7 Preparation of competent cells ……………………………………………...64 2.2.8 Transformation of plasmid into E. coli TOP10 competent cells…………….65 2.2.9 Transformation of plasmid into E. coli BL21 (DE3) competent cells…………………………………………………………… ...65 2.2.10 Long-term storage of recombinant plasmid…………………………………66 2.2.11 Protein analysis ……………………………………………………………..66 2.2.11.1 Determination of protein concentration …………………...66 2.2.12 Protein analysis by SDS-PAGE……………………………………………..68 2.2.13 Determination of immunogenicity of the expressed protein………………...69 2.2.13.1Electrophoretic transfer of proteins to membrane (Western blotting)…………………………………………..69 2.2.13.2Immunoreactivity study of the fusion proteins by Western blot analysis………………………………………70 2.2.13.2.1 Detection of histidine-tagged protein……...... 70 2.2.13.2.2 Detection of GST-tagged protein…………………70 2.2.13.2.3 Detection of specific IgG4 antibody………………70 2.2.13.2.4 Development of Western blot using chemiluminescence substrate……………………...71 2.2.14 Expression of the recombinant proteins……………………………………..71 2.2.15 Fast Protein Liquid Chromatography (FPLC) protein purification……….…72 2.2.15.1 Preparation of the AKTATMprime………………………….72 2.2.15.2 Stripping and recharging of the resin ……………………...73
CHAPTER 3: CLONING AND EXPRESSION OF ORF OF THE GENES ENCODING TES-26, TES- 32 AND TES-120 IN E. coli
3.1 Introduction…………………………………………………………………………..74
9 3.2 Methodology and results……………………………………………………………..78 3.2.1 Isolation of T. canis poly(A)+ RNA………………………………………...78 3.2.2 Oligonucleotides design..…………………………………………………....79 3.2.2.1 Mutagenic oligonucleotide primers…………………………………79 3.2.3 Construction of rTES-26, rTES-32 and rTES-120 recombinant plasmids……………………………………………………………………...82 3.2.3.1 RT-PCR of ORF of the genes encoding TES-26, TES-32 and TES-120……………………………………………….83 3.2.3.1.1 Purification of RT-PCR products………………….87 3.2.3.2 Cloning of RT-PCR products into PCR2.1 TOPO-TA vector ……..87 3.2.3.3 Verification of positive clones……………………………………...87 3.2.3.3.1 PCR screening of TOPO transformants…….……..87 3.2.3.3.2 Restriction endonuclease digestion of the recombinant plasmids……………………………..89 3.2.3.3.3 Confirmation by DNA sequencing………………..93 3.2.3.4 In-vitro mutagenesis……………………………………………….103 3.2.3.4.1 PCR-based site-directed mutagenesis……………103 3.2.3.5 Cloning into expression vectors……..…………………………….108 3.2.3.5.1 Vectors and inserts preparation…………………..108 3.2.3.5.1.1 Single digestion with restriction enzyme ………………………………….108 3.2.3.5.1.2 Dephosphorylation of vectors…………...115 3.2.3.5.1.3 Gel-purification of DNA inserts ……..... 115 3.2.3.5.2 Ligation of inserts into expression vectors………115 3.2.3.5.3 Analysis of pPROExHT/pET recombinants……..116 3.2.3.5.3.1 PCR screening…………………………...116 3.2.3.5.3.2 DNA sequencing…………………….…..120 3.2.3.5.4 Transformation of pPROExHT/pET recombinants into expression host…………….…120 3.2.4 Protein expression………………………………………………………….125 3.2.4.1 Small-scale expression…………………………………....125 3.2.4.1.1 Optimizing the expression of the rTES-26, rTES-32 and rTES-120 recombinant proteins……125 3.2.4.1.1.1 Induction of the target proteins at different induction time-point……………125 3.2.4.2 Determination of the target protein solubility and confirmation of presence of protein tags…………… .129 3.2.4.2.1 Determination of histidine-tagged in rTES-120 recombinant protein by Western blot analysis…...130 3.2.4.2.2 Determination of GST-tagged in the recombinant protein by Western blot analysis…………………………………………..….133
3.3 Discussion……………………………………………………………….………….136
CHAPTER 4 PURIFICATION OF rTES-26, rTES-32 AND rTES-120 RECOMBINANT PROTEINS IN E. coli
4.1 Introduction……………………………………………………………….………...141
4.2 Methodology 4.2.1 Purification of rTES-120 histidine-tagged recombinant protein………….. 143 4.2.1.1 Large scale protein expression………………………………….…143
10 4.2.1.2 Preparation of cleared cell lysates under native conditions………..143 4.2.1.3 Optimization of rTES-120 recombinant protein purification using FPLC under native conditions………………………………144 4.2.2 Purification of rTES-26 and rTES-32 GST-tagged recombinant proteins…………………………………………...………………..……….146 4.2.2.1 Large scale protein expression……………………………….……146 4.2.2.2 Cell extracts preparation for native purification…………….…….146 4.2.2.3 Laboratory scale purification of GST-tagged proteins under native conditions……………………….………………………..…147 4.2.2.4 Proteolytic cleavage of target protein from GST Tag………….….149 4.2.2.4.1 Small-scale optimization of proteolytic cleavage….…...149 4.2.2.4.2 Scale-up of cleavage reaction………………………..…150 4.2.2.4.3 Factor Xa capture after cleavage…………………….…150 4.2.3 Determination of immunoreactivity of the purified rTES-120 recombinant proteins by Western blot technique…………………….…..151 4.2.4 Determination of immunoreactivity of the cleaved rTES-26 and rTES-32 recombinant proteins by Western blot technique……………..….152
4.3 Results………………………………………………………………………………152 4.3.1 Optimization of rTES-120 recombinant protein purification using FPLC under native conditions…………………………………………..….152 4.3.2 Purification of rTES-26 and rTES-32 GST fusion proteins under native conditions………………………………………….…………….….156 4.3.3 Small-scale optimization of proteolytic cleavage………………...... 159 4.3.4 Determination of immunoreactivity of the purified and cleaved recombinant proteins by Western blot technique……………...…………..162
4.4 Discussion…………………………………………………………………………..166
CHAPTER 5 DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) USING rTES-26, rTES-32 AND rTES-120 RECOMBINANT ANTIGENS
5.1 Introduction…………………………………………………………………………174
5.2 Materials and methodology…………………………………………………………175 5.2.1 Total IgE-ELISA…………………………………………………………...175 5.2.2 Optimization of different parameters in rTES-ELISA……………………..175 5.2.2.1 Concentration of TES recombinant antigens……………………176 5.2.2.2 Dilutions of primary antibodies…………………………………176 5.2.2.3 Dilutions of secondary antibodies……………………………….176 5.2.3 Indirect enzyme-linked immunosorbent assay (ELISA)…………………...177 5.2.4 Determination of positive cut-off optical density value (COV)…………...178 5.2.5 Method validation …………………………………………………………179 5.2.5.1 Determination of sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV)….…179 5.2.5.2 Statistical analysis………………………………………...... 179
5.3 Results……………………………………………………………………………..…180 5.3.1 Determination of total IgE in sera from toxocariasis patients, various parasitic infections and healthy normals………………………………...... 180 5.3.2 Optimization of different parameters in rTES-ELISA………………….….186 5.3.3 Sensitivity and specificity analysis of recombinant antigens using
11 IgG1, IgG2, IgG3, IgE and IgM…………………………………………....196 5.3.4 Sensitivity and specificity analysis of recombinant antigens using IgG4-ELISA………………………………………………………………..204 5.3.5 Statistical analysis……………………………………………….………....214
5.4 Discussion…………………………………………………………………………..216
CHAPTER 6 SUMMARY AND CONCLUSION…………………………………….…………………224
REFERENCES…………………………………………………………………………….233
APPENDICES Appendix A List of chemicals and reagents
Appendix B Complete cds sequences from the Genbank
LIST OF PUBLICATIONS, PRESENTATIONS AND PATENT
LIST OF TABLES Page no.
Table 2.1 E. coli strains employed in this study………………………………………..43
Table 2.2 Preparation of BSA standards………………………………………………..68
Table 3.1 The details of the primers used in this study………………………………...81
Table 3.2 The expected PCR product size for ORF of each gene……………………...82
Table 3.3 Primers used for repairing base-errors in site-directed mutagenesis……….105
Table 4.1 Concentrations of purified rTES-120 recombinant protein obtained by FPLC using different concentrations of salt and washing column volume……………………………………………………………………...153
Table 4.2 Concentrations of purified rTES-26 and rTES-32 recombinant proteins…..157
Table 5.1 Results of the screening of various categories of serum samples using Total IgE-ELISA (Total IgE-ELISA kit, IBL Hamburg, Germany)……………………………………………………………….…..181
Table 5.2 Summary of the results of the screening of total IgE in sera from toxocariasis patients, various parasitic infections and healthy normals (summarized from table 5.1)………………………………………………185
Table 5.3a Optimization of rTES-26 recombinant antigen concentration and serum dilution in IgG4-ELISA…………………………………………….187
12 Table 5.3b Optimization of rTES-32 recombinant antigen concentration and serum dilution in IgG4-ELISA………………………………………….…187
Table 5.3c Optimization of rTES-120 recombinant antigen concentration and serum dilution in IgG4-ELISA…………………………………………….188
Table 5.3d Optimization of the dilutions of IgG4 antibody conjugated to HRP in IgG4-ELISA………………………….………………………………....189
Table 5.4 Optimization of antigen concentrations and conjugate dilutions in IgG1-ELISA…………………………………………..…………………...190
Table 5.5 Optimization of antigen concentrations and conjugate dilutions in IgG2-ELISA………………..……………………………………………...191
Table 5.6 Optimization of antigen concentrations and conjugate dilutions in IgG3-ELISA……………………..………………………………………...192
Table 5.7 Optimization of antigen concentrations and conjugate dilutions in IgE-ELISA…………………………………………………………………193
Table 5.8 Optimization of antigen concentrations and conjugate dilutions in IgM ELISA…………………………………………………………………194
Table 5.9 Optimum conditions for each rTES-ELISA assay………………………....195
Table 5.10a Sensitivity and specificity analysis of rTES-26 ELISA……………………197
Table 5.10b Sensitivity and specificity analysis of rTES-32 ELISA…………………....199 Table 5.10c Sensitivity and specificity analysis of rTES-120 ELISA………………..…201
Table 5.11 Sensitivity and cross-reaction of sera from patients with other parasitic infections in rTES-ELISA using IgG subclasses, IgE and IgM…………… 202 Table 5.12 Sensitivity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens………………………………………………...206
Table 5.13 Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens………………………………………………...207
Table 5.14 Summary of sensitivity evaluations of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM………….………………………...212
Table 5.15 Summary of specificity evaluations of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM……………………..……………..212
Table 5.16a Specificity, accuracy, PPV and NPV of rTES ELISA using monoclonal mouse anti-human IgG4 antibody (include 30 healthy normals) ……………………………………………………………..……..213 Table 5.16b Specificity, accuracy, PPV and NPV of rTES ELISA using monoclonal mouse anti-human IgG4 antibody (exclude healthy normals)
13 ……………………………………………..……………………..213
Table 5.17 One-Way ANOVA analysis of the comparison of mean O.Ds of IgG4 assays on 30 toxocariasis serum samples using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens………..…………………215
Table 5.18 Pearson Chi-Square analysis of the comparison of specificity of IgG4 assays using rTES-26, rTES-32, rTES-30USM and rTES-120 antigens…………………………………………………………………….215
LIST OF FIGURES Page no.
Figure 1.1a: Adult male (10 cm) and female worms (18 cm) obtained from small intestine of infected dog...... 4
Figure 1.1b: T. canis adult worm...... 4
Figure 1.1c: T. cati adult worm...... 4
Figure 1.2a: Unembryonated T. canis egg...... 5
Figure 1.2b: Infective egg containing L2 larva……………………………………….…..5
Figure 1.3: The summary of life cycle of T. canis…………………………………………...8
Figure 2.1 Modified Baermann’s apparatus for collection of T. canis larvae…………60
Figure 3.1 Strategy for cloning and expression of ORF of T. canis genes………76 & 77
Figure 3.2a RT-PCR product of ORF of the gene encoding TES-26 resolved on 1% DNA agarose gel electrophoresis……………..……………………..…84
Figure 3.2b RT-PCR product of ORF of the gene encoding TES-32 resolved on 1% DNA agarose gel electrophoresis…………..…………………………..85
Figure 3.2c RT-PCR product of ORF of the gene encoding TES-120 resolved on 1% DNA agarose gel electrophoresis……….……………………………..86
14 Figure 3.3 Map and sequence characteristics of PCR2.1-TOPO vector shows the cloning region…………………………………………………………88
Figure 3.4a PCR product of TOPO/TES-26 transformants resolved on 1% DNA agarose gel electrophoresis………..………………………………...90
Figure 3.4b PCR product of TOPO/TES-32 transformants resolved on 1% DNA agarose gel electrophoresis……………..…………………………91
Figure 3.4c PCR screening of TOPO/TES-120 transformants resolved on 1% agarose electrophoresis…………………………………………………...92
Figure 3.5a Restriction enzyme analysis of the TOPO/TES-26 construct resolved on 1% agarose gel electrophoresis……………………………...94
Figure 3.5b Restriction enzyme analysis of the TOPO/TES-32 constructs resolved on 1% agarose gel electrophoresis……..……………………….95
Figure 3.5c Restriction enzyme analysis of the TOPO/TES-120 constructs resolved on 1% agarose gel electrophoresis………..…………………….96
Figure 3.6a Contig alignment analysis of TOPO/TES-26#1and previously published sequence of TES-26……………..………………………….…97
Figure 3.6b Contig alignment analysis of TOPO/TES-32 and previously published sequence of TES-32……………………………………..…….99
Figure 3.6c Contig alignment analysis of TOPO/TES-120 and previously published sequence of TES-120………………………………………….101
Figure 3.7 Schematic overview of the Quickchange® site-directed mutagenesis method (Stratagene, USA)……………………………...….104
Figure 3.8 Agarose gel electrophoresis analysis of the site-directed mutagenesis product of TOPO/TES-26…………………...……………...106
Figure 3.9a Map of pET-42a-c vector shows the multiple cloning regions…….…….109 Figure 3.9b Sequence characteristics of pET-42a-c vector shows the cloning and expression regions………………………..………………………….110
Figure 3.10 Map and multiple cloning sites of pPROExTMHT expression vectors…...111
Figure 3.11a Restriction enzyme analysis of TOPO/TES-26 and pET42b resolved on 1% DNA agarose gel electrophoresis……………………….111
Figure 3.11b Restriction enzyme analysis of TOPO/TES-32 and pET42a resolved on 1% DNA agarose gel electrophoresis………………………………….111
Figure 3.11c Restriction enzyme analysis of TOPO/TES-120 and pPROExTMHTa resolved on 1% DNA agarose gel electrophoresis………………………..112
Figure 3.12a PCR screening of pET/TES-26 recombinants resolved on 1% agarose electrophoresis……………………………………………………115
Figure 3.12b PCR screening of pET/32 recombinants resolved on 1% agarose
15 electrophoresis……………………………………………………………116
Figure 3.12c PCR screening of pPROExTMHT recombinants resolved on 1% agarose electrophoresis………..…………………………………………117
Figure 3.13a Amino acid sequence of pET42/TES-26 construct………………………119
Figure 3.13b Amino acid sequence of pET42/TES-32 construct……………………....122
Figure 3.13c Amino acid sequences of pPROExTMHTa /TES-120 construct………….124
Figure 3.14a SDS-PAGE analysis of rTES-26 recombinant protein at different post-induction time-point………………………………………………..126
Figure 3.14b SDS-PAGE analysis of rTES-32 recombinant protein at different post-induction time-point………………………………………………..127
Figure 3.14c SDS-PAGE analysis of rTES-120 recombinant protein at different post-induction time-point………………………………………………..128
Figure 3.15 Western blot analysis of rTES-120 recombinant protein expressed in supernatant and pellet…………………………………………………132
Figure 3.16 Western blot analysis of rTES-26 recombinant protein expressed in supernatant and pellet………………………………………………….134
Figure 3.17 Western blot analysis of rTES-32 recombinant protein expressed in supernatant and pellet………………………………………………….135
Figure 4.1 Overview strategies for purification of the T. canis recombinant proteins under native conditions………………..…………………..……142
Figure 4.2 FPLC protein purification was performed using AKTATMprime (Amersham BioSciences, Sweden)……….………………………………145
Figure 4.3 Purification of rTES-26 and rTES-32 GST-tagged proteins using gravity flow…………………………………...………………………….148
Figure 4.4 The output chromatogram result of purification of rTES-120 recombinant protein using FPLC method…………………...…………...154
Figure 4.5 SDS-PAGE analysis of rTES-120 protein fractions collected using FPLC method…………………………………………………..……….. 155
Figure 4.6 SDS-PAGE analyses of rTES-26 and rTES-32 GST fusion protein fractions…………………………………………………………………..158
Figure 4.7 SDS-PAGE analysis of rTES-26 protein after cleavage at different enzyme : protein ratio and incubation times…………………………..…160
Figure 4.8 SDS-PAGE analysis of rTES-32 protein after cleavage at different enzyme : protein ratio and incubation times……………………….….…161
Figure 4.9 Western blot analysis showing the immunogenicity and specificity of the purified rTES-120 recombinant protein……………………….…164
16 Figure 4.10 Western blot analysis showing the immunogenicity and specificity of the cleaved rTES- 26 recombinant protein…………….……………..165
Figure 4.11 Western blot analysis showing the immunogenicity and specificity of the cleaved rTES-32 recombinant protein…………..……………..…166
LIST OF ABBREVIATIONS & ACRONYMS
µg microgram µl microliter ABTS 2,2’-Azino-bis(3-ethylbenthiazoline-6- sulphonic acid) AEC absolute eosinophil counts AP ammonium persulfate BM Boehringer Mannheim bp base pair CFT complement fixation test CIEP countercurrent immunoelectrophoresis CT covert toxocariasis CV column volume DEC diethylcarbamazine DEPC diethyl pyrocarbonate DFAT direct fluorescent antibody tests DGDT double gel diffusion test DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EME eosinophilic meningo-encephalitis ES excretory secretory FPLC Fast Protein Liquid Chromatography GST glutathione S-transferase GWC Goldmann-Witmer coefficient HRP horseradish peroxidase HUKM Hospital Universiti Kebangsaan Malaysia HUSM Hospital Universiti Sains Malaysia IACE indirect antibody competition ELISA ICT immunochromatography test IFAT indirect fluorescent antibody test IL interleukin
17 IPTG isopropyl- ß-D- thiogalactopyranoside K2HPO4 dipotassium hydrogen phosphate kb kilobase kDa kilo dalton KH2PO4 potassium dihydrogen phosphate L2 stage two larva LB Luria-Bertani MCS multiple cloning site mg milligram MgCl2 magnesium chloride MRI magnetic resonance imaging mRNA messenger ribonucleic acid MtDNA mitochondrial DNA N2 nitrogen Na2HPO4 disodium hydrogen phosphate NaH2PO4 sodium dihydrogen phosphate NaHCO3 sodium bicarbonate Ni2+ níckel ion NIH National Institutes of Health Ni-NTA nickel - nitrilo-tri-acetic acid nm nanometer NPV negative predictive value NS nephrotic syndrome NTD neglected tropical diseases OD optical density ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS Phosphate buffered saline PBS-T PBS-Tween 20 PCR polymerase chain reaction pmol picomole POD peroxidase PPV positive predictive value PRIST paper radio-immunosorbent test RAPD random amplification of polymorphic DNA RAST radio-allergosorbent test rDNA ribosomal DNA RFLP restriction fragment length polymorphism RIA radio immunoassay RNA ribonucleic acid rpm revolution per minute RPMI Roswell Park Memorial Institute rTES recombinant Toxocara excretory-secretory RT-PCR reverse-transcriptase polymerase chain reaction SAP shrimp alkaline phosphatase SDS sodium dodecyl sulphate SOC Super Optimal broth with Catabolite repression STH Soil-transmitted helminthiases TB Terrific Broth TBE Tris-Borate EDTA TBS Tris buffered saline TBS-T TBS-Tween 20 TBSTT TBS/Tween 20/Triton-X TEMED N,N,N',N'-Tetramethylethylenediamine TES Toxocara excretory-secretory
18 Th2 T helper 2 TMB 3,3’,5,5’-tetramethylbenzidine USB ultrasound biomicroscopy UV ultra violet VLM visceral larva migrans WB Western blot
PENGKLONAN, PENGEKSPRESIAN DAN PENULENAN ANTIGEN REKOMBINAN Toxocara canis (rTES-26, rTES-32, rTES-120) DAN PEMBANGUNAN UJIAN SERODIAGNOSTIK BAGI TOKSOKARIASIS
Abstrak
Serodiagnosis rutin untuk penyakit toksokariasis pada manusia adalah berdasarkan kit
ELISA-IgG tak-langsung yang menggunakan antigen ekskretori-sekretori natif daripada
Toxocara canis. Walau bagaimanapun, asai tersebut mempunyai spesifisiti yang rendah, terutamanya untuk kegunaan di negara tropika yang mempunyai pelbagai jangkitan parasit.
Dalam usaha untuk meningkatkan kejituan ujian diagnostik bagi penyakit ini, asai ELISA-
IgG4 telah dibangunkan dengan menggunakan tiga antigen rekombinan.
Dalam kajian ini, DNA rekombinan T. canis yang mengkodkan antigen rekombinan rTES-26, rTES-32 dan rTES-120 dihasilkan dengan mengklonkan kerangka bacaan terbuka gen masing-masing melalui kaedah “reverse-transcriptase-PCR” (RT-PCR) menggunakan mRNA yang diekstrak daripada kultur larva T. canis peringkat kedua ke dalam vektor
PCR2.1 TOPO. Analisis jujukan menunjukkan TOPO/TES-32 and TOPO/TES-120 mempunyai persamaan 100% dengan jujukan yang dilaporkan dalam “GenBank”, tetapi fragmen gen TOPO/TES-26 mempunyai empat mutasi. Selepas kesemua mutasi dalam
TOPO/TES-26 diperbaiki, TES-26 dan TES-32 kemudiannya disubklonkan ke dalam vektor
19 ekspresi yang mempunyai penanda GST, manakala TES-120 disubklonkan ke dalam vektor yang mempunyai penanda histidin, dan kesemuanya diekspreskan di dalam hos ekspresi E. coli BL21(DE3).
Protein rekombinan tersebut kemudiannya ditulenkan secara natif dengan kaedah kromatografi afiniti menggunakan resin GST dan His-Trap kerana protein rekombinan tersebut banyak terhasil dalam bentuk yang terlarut. Protease tapak spesifik, Factor Xa, digunakan untuk membuang penanda GST dalam protein rekombinan TES-26 (rTES-26) dan
TES-32 (rTES-32). Analisis blot Western menunjukkan antigen rekombinan tersebut adalah reaktif dan spesifik secara imunologik. Serum daripada pesakit toksokariasis yang mempunyai antibodi IgG4 dapat mengenalpasti antigen rekombinan tersebut, manakala serum daripada pesakit jangkitan lain dan individu sihat adalah tidak reaktif.
Apabila ketiga-tiga antigen rekombinan diuji dengan ELISA yang spesifik untuk immunoglobulin klas IgM dan IgE dan subklas IgG (IgG1-IgG4), keputusan jelas menunjukkan bahawa hanya asai IgG4 memberikan spesifisiti yang baik. Keupayaan diagnostik setiap antigen rekombinan tulen dan rTES-30USM (dihasilkan sebelum ini di dalam makmal kami) seterusnya dinilai dengan asai ELISA-IgG4 menggunakan sampel serum sebanyak 242 yang termasuk 30 pesakit toksokariasis dengan bukti klinikal, hematologi dan serologi. Kedua-dua asai rTES-26 dan rTES-32 ELISA menunjukkan sensitiviti 80.0%; manakala rTES-120 ELISA-IgG4 menunjukkan sensitiviti 93.3.0%, sama seperti yang telah dilaporkan sebelum ini untuk rTES-30USM ELISA-IgG4. Tahap sensitiviti rTES-120/rTES-30USM ELISA-IgG4 adalah lebih tinggi secara signifikan daripada rTES-26/ rTES-32 ELISA-IgG4 (p<0.001). Walau bagaimanapun, perbandingan min O.D 30 sampel toksokariasis di antara asai IgG4 dengan menggunakan empat antigen recombinan tidak menunjukkan perbezaan yang signifikan (p>0.05). Pada tahap signifikan p<0.05, tiada perbezaan yang signifikan di antara spesifisiti rTES-26 dan rTES-120
(p=0.059), di antara rTES-26 dan rTES-30USM atau di antara rTES-30USM dan rTES-120.
20 Dalam asai terakhir, rTES-32 tidak dimasukkan kerana ia tidak menunjukkan sensitiviti dan spesifisiti yang lebih baik daripada rTES-26. Malah rTES-30USM dimasukkan kerana sensitivitinya yang tinggi dan pengesanan 100% kes toksokariasis dapat dicapai apabila keputusan asai IgG4 menggunakan rTES-30USM dan rTES-120 digabungkan.
Kesimpulannya, asai terakhir yang sensitif (80.0% - 93.3%) dan spesifik (92.0%
-96.2%) untuk pengesanan penyakit toksokariasis telah berjaya dibangunkan dengan menggunakan tiga telaga yang bersebelahan, setiap satunya disalut dengan rTES-26, rTES-
30USM dan rTES-120. Kajian ini ádalah novel dalam beberapa aspek iaitu ia adalah yang pertama melaporkan penggunaan pelbagai antigen rekombinan untuk serodiagnosis toksokariasis; penggunaan rTES-26 (dan rTES-32) dalam serodiagnosis jangkitan toksokara; penggunaan asai IgG4 untuk rTES-120 dan rTES-26 dan penggunaan penanda GST dalam ekspresi dan penulenan protein recombinan toksokara. Ujian ini mungkin merupakan pembaharuan yang signifikan berbanding ujian komersial yang ada bagi diagnosis penyakit toksokariasis, terutamanya bagi negara yang ko-endemik dengan cacing tularan tanah yang lain.
21 CLONING, EXPRESSION AND PURIFICATION OF Toxocara canis RECOMBINANT ANTIGENS (rTES-26, rTES-32, rTES-120) AND DEVELOPMENT OF SERODIAGNOSTIC TEST FOR TOXOCARIASIS
Abstract
Routine serodiagnosis of human toxocariasis is based on indirect IgG-ELISA kits which employ native Toxocara canis excretory-secretory (TES) antigen. However, these assays lacked specificity especially when used in tropical countries where multiparasitism are prevalent. In an effort to improve the diagnostic test for this infection, we have developed an
IgG4-ELISA assay which uses three recombinant antigens.
In this study, recombinant T. canis DNA which encode for rTES-26, rTES-32 and rTES-120 were produced by cloning of open-reading frames (ORF) of the respective genes via reverse-transcriptase-PCR (RT-PCR) using mRNA extracted from a culture of T. canis second stage larvae into PCR2.1 TOPO vector. Sequence analysis revealed that TOPO/TES-
32 and TOPO/TES-120 were 100% similar to the reported sequences in the GenBank, however, TOPO/TES-26 gene fragment had four mutations. After all mutations in
TOPO/TES-26 gene fragments had been corrected, TES-26 and TES-32 were subsequently subcloned into a GST-tagged, while TES-120 was subcloned into a His-tagged prokaryotic
22 expression vector; and all constructs were expressed in E. coli BL21(DE3) expression host.
The recombinant proteins were subsequently purified under native condition by affinity chromatography using GST and His-Trap resins since these recombinant proteins were abundantly expressed in soluble form. The site-specific protease, Factor Xa, was used to remove GST tag in the TES-26 and TES-32 GST-tagged fusion proteins. Western blot analysis revealed that these recombinant antigens were immunologically reactive and specific. Sera from patients infected with toxocariasis had IgG4 antibodies that recognized these recombinant antigens, while sera from individuals with other infections and from healthy normals did not.
When the three recombinant antigens were tested in ELISAs specific for immunoglobulin IgM and IgE classes, and IgG subclasses (IgG1-IgG4), the results clearly showed that only IgG4 assay displayed good specificity. The diagnostic utility of each purified recombinant antigen and rTES-30USM (previously produced in our laboratory) was further evaluated by IgG4-ELISA assay using 242 serum samples which included 30 sera from patients with clinical, haematological and serological evidence of toxocariasis. Both rTES-26 and rTES-32 IgG4-ELISAs demonstrated sensitivity of 80.0%; while rTES-120
IgG4-ELISA showed sensitivity of 93.3.0%, which is similar to that previously reported for rTES-30USM IgG4-ELISA. The sensitivity of rTES-120/rTES-30USM IgG4-ELISA was found to be significantly higher than rTES-26/rTES-32 IgG4-ELISA (p<0.001). However, the mean O.Ds of the 30 toxocariasis samples among the IgG4 assays using the four recombinant antigens were shown not to be significantly different (p>0.05). At p<0.05, there was marginally no significant difference between the specificities of rTES-26 and rTES-120 (p=0.059), rTES-26 and rTES-30USM or between rTES-30USM and rTES-120.
In the final assay, rTES-32 was excluded since it was not better than rTES-26 in terms of sensitivity or specificity. Instead rTES-30USM was included due to its high sensitivity and the fact that a 100% detection of toxocariasis cases was achieved when results of IgG4 assays using rTES-30USM and rTES-120 were combined.
23 In summary, a final assay which is sensitive (80% to 93.3%) and specific (92.0%-
96.2%) for detection of toxocariasis was successfully developed using three adjacent wells, each separately coated with rTES-26, rTES-30USM and rTES-120. This study is novel in several ways namely it is the first report of the use of multiple recombinant antigens for serodiagnosis of toxocariasis; the use of rTES-26 (and rTES-32) in Toxocara serodiagnosis; the use of IgG4 assay for rTES-120 and rTES-26 and the use of GST tag in the expression and purification of Toxocara recombinant proteins. These test maybe a significant improvement over commercially available tests for diagnosis of toxocariasis and may be use especially in countries co-endemic with other soil-transmitted helminthes.
CHAPTER 1
INTRODUCTION
1.1 General introduction
Human toxocariasis is a worldwide parasitic zoonosis, caused most commonly by the parasite dog intestinal roundworm (Toxocara canis) and less frequently the cat roundworm
(Toxocara cati) (Despommier, 2003; Fisher, 2003). Although several nematodes have been reported to produce visceral larva migrans (VLM), T. canis appears to be the primary causative agent (Glickman & Schantz, 1981). T. canis is more important in causing human infection than T. cati because dogs are more often in direct contact with people than cats. In addition cats usually select sandy soil for defecation and habitually bury Toxocara eggs- contaminated faeces, making infectious eggs less accessible to susceptible individuals
(Overgaauw, 1997; Smith & Rahmah, 2006). Nevertheless, T. cati has been implicated particularly in ocular toxocariasis and represents an underestimated zoonotic agent (Fisher,
2003). Human toxocariasis is still a poorly diagnosed disease, especially in places with low socioeconomic level, and is largely unknown to health professionals and the general population (Wells, 2007). Nevertheless, it is probably one of the most common zoonotic helminthiases in temperate climates (Schantz, 1989).
24 Toxocariasis is known to be a disease prevalent among preschool children since they play with dogs on open grounds and thus have a higher chance to ingest eggs-contaminated soil. Since Toxocara infection is commonly underdiagnosed, it may be thought to be an insignificant cause of morbidity in populations (Park et al., 2000; Rahmah et al., 2005). In
Malaysia, the perception that toxocariasis is not of public health importance is probably due to scarcity of reports on Toxocara seroprevalence (Lokman Hakim et al., 1992, 1993, 1997;
Patrick et al., 2001) and the unsatisfactory specificity of the test employed (Smith &
Rahmah, 2006).
1.2 The taxonomy of Toxocara and morpholyogy
The genus Toxocara belongs to the phylum nemathelminthes, class nematode, family
Toxocaridae, superfamily Ascaridoidea and order Ascaridida (Despommier, 2003). There are few reported studies on species other than T. canis and T. cati such as T. vitulorum (bovids) and T. pteropodis (nematode of fruit bats). Larvae of T. vitulorum and Toxascaris leonine can invade the tissues of laboratory animals, but to what extents they are implicated in human disease are uncertain (Thompson, 1994).
Several species of Toxocara infecting domestic cats and other felids have been identified namely T. malayasiensis which infect domestic cats in Malaysia and China
(Gibbons et al., 2001; Li et al., 2006) and T. lyncus in caracals (related to lynxes). However, it is unknown whether these two newly described organisms cause human infection resulting in clinical disease (Zhu et al., 1998). Other Toxocara species which are found in rodents and other wild animals, with no reported links to disease in humans or domestic animals include
T. tanuki, T. alienata and T. mackerrasae.
There are other ascaridids that can invade human tissues. For example, larvae of
Baylisascaris procyonis (common intestinal ascarid of raccoon, Procyon lotor) were identified as the causative agent in young patients with severe and fatal eosinophilic meningoencephalitis (Fox et al., 1985). As with Toxocara spp., transmission to humans
25 occurs directly through ingestion of embryonated eggs from the soil or from contaminated hands. Ascaris is also one of the public health importances in many countries but it is not usually considered an aetiology agent for VLM in humans. However, there are suggestions that Ascaris suum can cause VLM in humans in Japan (Sakakibara et al., 2002).
Traditionally, ascaridid species have been identified based on morphological features. Morphological observations are unable to differentiate T. malayasiensis from other ascaridoid species, and this had caused confusion regarding the identity of "T. canis" from cats in Malaysia. This "T. canis" from cats in Malaysia was eventually proven to represent T. malayasiensis (Gibbons et al., 2001), which is genetically more similar to T. cati than T. canis (Zhu et al., 1998). The determination of specific identity of ascaridoid larvae recovered from animal or human tissue is almost impossible because they are small (less than 0.5 mm long). Approaches such as karyotyping and electrophoretic techniques, DNA approaches such as restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR) and random amplification of polymorphic DNA (RAPD) have also been applied for studying the taxonomy of the members within the genus Toxocara (Wu et al., 1997; Epe et al., 1999;
Zhu et al., 2001; Li et al., 2007).
The T. canis adult female worm is 5 to 18 cm long and the male worm is 4 to 10 cm
(figure 1.1a). T. canis and T. cati are easily be distinguished by the shape of cervical alae
(figures 1.1b and 1.1c) or by the species of host animals from which they are recovered. T. canis adult worm is identified based on the presence of an oesophageal ventriculus and spear-shaped cervical alae. This parasite differs from T. cati, which possesses arrow-shaped cervical alae (Uga et al., 2000).
It has also been shown that the two species of eggs can be distinguished by their surface structure. The measurement of egg size is not a good criterion to differentiate T. canis and T. cati eggs, since both species of eggs are similar in size and morphology. Even though T. canis eggs tend to be relatively larger than T. cati eggs, this feature is not adequate for differentiation. The T. canis egg is almost spherical and about 90 x 75 µm with a light
26 brown thick shell that is pitted and coarser than T. cati (figures 1.2a and 1.2b). The infective larva is approximately 400 x 20 µm and resembles the adult worm.
1.3 The larval surface coat
T. canis bears an extracellular cuticle which covers the whole body and lying outside the external cell layer is the hypodermal or epidermal syncytium. The cuticle is on an average
300 - 400 nm thick. The external surface coat of the T. canis larva is covered by a carbohydrate-rich glycocalyx called the surface coat, which is a fuzzy envelope 10 nm in thickness and is detached by a similar distance from the epicuticle (Page et al., 1992b).
Figure 1.1a Adult male (10 cm) and female worms (18 cm) obtained from small intestine of infected dog
spear-shape arrow-shape cervical alae cervical alae
27
Figure 1.1b T. canis adult worm Figure 1.1c T. cati adult worm
(Reference : http//www.vetsci.psu.edu/coursedesc/vsc402/27ascarids.htm)
28 The T.canis adult female worm is 5 to 18 cm long and the male worm is 4 to 10 cm.
The adults live in the lu men of the small intestine. The egg is about 85 mm 75 mm with a
light brown and thick she ll that is pitted (figure 1.1a and 1.1b). The infective larvae is
approximately 400 mm 20 mm and resembles the adult worm.
Figure 1.2a : Unembryonated T.canis egg
FigureFigu 1.2bre 1.2b : Infective : Infective egg egg containing containin L2g L2 larva larva
(Reference: http//www.standford.edu/class/humbiolo3/Parasites2005/Toxocariasis/parasite%20.htm)
29 Biochemical analysis of the surface coat of infective larvae, coupled with electron microscopy, showed that its principal constituent is a labile antigenic coat that consists primarily of TES-120, a set of secreted mucins which is also a dominant secreted product.
The surface coat carries a strong negative charge, binding to cationic stains such as ruthenium red and catinized ferritin (Page et al., 1992a), but the identity of this charge group has yet to be determined.
T. canis larvae alter their surfaces rapidly after they hatch from their eggs. The surface coat is frequently the target of high-level antibody responses by the infected humans.
It is rapidly shed when the parasite is under immune attack along with adherent effector cells and antibodies, providing T. canis with a dramatic means of evading immune destruction
(Page et al., 1992b). After hatching, larvae initiate expression of TES-32 and TES-120 surface antigens and also initiate secretion of these proteins in large quantities. Other proteins such as TES-70 and TES-400 are also secreted (Maizels et al., 1987). The antibody binds to the coat resulting in the loss of the surface coat. This extracellular surface coat has been demonstrated on T. canis larvae prior to hatching and during all stages of in vitro culture, suggesting that it may play a role in protecting the parasite on hatching in the gastrointestinal tract (Page et al., 1992b).
1.4 Toxocara excretory/secretory antigens (TES)
Larvae secrete a complex set of glycoproteins namely TES products in culture (de Savigny,
1975). They are classified according to their molecular weight on SDS-PAGE. Within TES is a group of highly immunogenic mucins which coat the parasite's surface and are shed into the host (Loukas et al., 2000a; Doeden et al., 2001).
In 1975, de Savigny has successfully cultured T. canis second-stage larvae in vitro for prolonged periods. The amount of TES produced by an individual larva in culture is between 9 pg and 8 ng of protein per day. TES are the first mosaic of antigens to which an antibody response is detectable following infection and was used as specific target antigen for serodiagnosis in humans (de Savigny, 1975). Currently, TES products have been
30 extensively used in the diagnosis of human toxocariasis especially in antibody detection assay. The use of TES products in simple ELISA provided a sensitive and specific test, although it showed cross-reaction with other nematode species. The disadvantage of TES is that its production is limited to the capacity of parasite culture (Smith & Rahmah, 2006).
1.5 Life cycle of T. canis
The life cycle of T. canis is complex and is summarized in Figure 1.3. Dogs and other canids are the definitive hosts for T. canis. Puppies younger than 10 weeks of life are typical reservoirs and main shedders of T. canis, due to prenatal (transuterine) and/or transcolostral
(lactogenic) transmissions (vertical larval transmission). Almost all puppies are infected at or soon after birth (Overgaauw, 1997). Dogs may also be infected by consuming paratenic hosts (such as rodents) because of their predatory habits. Transfer of infective stage larvae to other animals through predation is a common mode of transmission among carnivorous vertebrates and this type of parasite transfer can occur from animals to humans
(Fan et al., 2004; Taira et al., 2004). Swallowing of infective eggs by adult dog rarely results in the presence of adult Toxocara worms in the digestive tract.
In non-pregnant adult dogs more than 5 weeks old, when the embryonated eggs are swallowed, they hatch in the small intestine and release larvae. Majority of the juvenile worms (larvae) penetrate the intestinal mucosa, enter the general circulation via blood and lymph and migrate to the liver and lungs, and are disseminated by the systemic circulation
(somatic migration). Eventually, these larvae penetrate through capillary vessels and migrate to surrounding tissues preferentially in muscles, kidneys, liver and central nervous system where they may survive for years without undergoing further development
("hypobiosis") and remain as somatic larvae. This phenomenon also occurs in paratenic hosts. Only in a proportion of adult dogs some larvae undergo tracheal migration resulting in intestinal infection (Despommier, 2003).
31 Main spread of infection Less important spread of infection
Figure 1.3 The summary of life cycle of T. canis
(Reference: www.dpd.cdc.gov/ Toxocara_canis _lifecycle )
In pregnant and lactating dogs, these hypobiotic or dormant somatic larvae are released and reactivated by hormonal stimuli, where they migrate transplacentally into the
32 organs of the fetus as a vertical infection. Toxocara also infects newborn puppies through lactogenically transmitted larvae. This is why newborn puppies are likely to be infected. The tracheal migration pattern occurs via the liver, lungs and trachea leads to the development of adult and patent worms in the intestine. This liver-lung-trachea-oesophagus route is mainly attributed to young animals. Through swallowing, the larvae mature rapidly into reproductive adult worms in the intestinal tract of infected dogs. The adult female worms begin to produce a large number of eggs, up to 200,000 per day that contaminate the environment through dog faeces. The eggs are then ingested by final and paratenic hosts especially children (Despommier, 2003).
T. canis eggs are unembryonated when voided in the faeces of dogs into the environment, and therefore not infective to paratenic hosts. It requires a certain period of embryonation to develop into infective stage. Larvae moult once inside the eggs to become infective second-stage larvae. The eggs develop into embryonated eggs that are infectious to the final hosts and paratenic hosts under optimal temperatures and humidity. The eggs take
54 days to become infective at 12-18ºC, but only 14 days at 25-30ºC (Despommier, 2003).
Humans act as paratenic hosts in which Toxocara larvae do not develop beyond the second larval stage and never develop into adult worms. Children are at the highest risk of infection because of pica, geophagia and playing on soils contaminated with infected dog faeces. There are various routes of infection by Toxocara species. Humans and other paratenic hosts (such as rodents and rabbits) become infected by accidentally ingesting
(swallowing) embryonated Toxocara eggs containing infective larvae (faecal-oral route transmission) in soil, which is the major source of human toxocariasis. Other routes are by direct contact with infective soil or indirectly by eating food contaminated with infective soil
(sapro-zoonosis) such as unwashed raw vegetables. The potential for transmission of helminth eggs by unwashed vegetables (such as salad and carrots) were documented by previous study (Kozan et al., 2005).
Infections can also occur following the ingestion of infective larvae in undercooked meats from paratenic hosts such as chicken, cattle, duck, sheep and rabbits (Glickman &
33 Schantz, 1981; Struchler et al., 1990; Hoffmeister et al., 2007). Cases of adult hepatic toxocariasis have recently been increasingly reported and this trend may be attributed to the habit of eating uncooked animal tissue by adults (Morimatsu et al., 2006). This suggested that infective larvae can be released from animal tissues during digestion and subsequently cause human toxocariasis. Transmission following dispersion of eggs by filth flies has been reported since flies ingest 1-3 mg faeces over 2 - 3 hours. Toxocara eggs have been found on
2.4% of wild-caught naturally infected flies in Nigeria (Umeche & Mandah, 1989).
However, Toxocara eggs require a certain period of embryonation before being infective, although flies could deposit eggs on material that could be ingested later.
Toxocariasis cannot be spread from person to person. Humans also cannot be infected through direct contact with infected dogs or cats faeces because recently shed, unembryonated Toxocara eggs need to mature in soil for a minimum of 3 weeks before they become infective; however, Wolf and Wright (2003) reported that soil contamination is not the only effective route of transmission in human toxocariasis. T. canis eggs have been found in dog and fox hairs (Roddie et al., 2008) and consequently are a potential transmission route in humans. Aydenizoz-Ozkayhan et al. (2008) investigated the T. canis eggs in coats of different dog breeds as a potential transmission route in human toxocariasis.
Another route of transmission is by inhalation of infective windborne eggs following inhalations from aerosols and waterborne transmission by accidental ingestion during immersion watersports (Beer et al., 1999). Transmission to the fetus is possible in some paratenic hosts. In mice, T. canis and T. cati can infect the fetus if the mother is infected during pregnancy, but hypobiotic larvae from the tissues do not infect the fetus. However, vertical transmission in pregnant women does not occur. Transmission is also possible via milk in rodents (Overgaauw, 1997).
Upon hatching in the human small intestine, the emerging larvae cannot complete their normal development. The immature larvae enter the circulatory system and undergo somatic migration for months through soft tissues of the body such as liver, lungs, muscles, neurological tissues and various organs, but failed to reach maturity. They remain for many
34 years in an arrested state without growth, differentiation or reproduction until they are recognized by human immune system which gives rise to inflammatory reaction and finally death. Toxocara larvae can survive in human tissues for at least 9 years and possibly, for the entire life of the host.
1.6 Egg survival
Toxocara species eggs have thick, strong and complex shell layers to protect them from environmental factors such as direct sunlight and chemical agents. After 2-5 weeks, in conditions of suitable temperature (18-35ºC), relative humidity higher than 85%, and oxygenation, the infective stage (L2) develops inside the egg. They can survive in the soil for several years under appropriate temperature, humidity and oxygen conditions, becoming the source of infection to definitive and paratenic hosts. However, the infectivity of the embryonated eggs that were kept in 2% formalin at 28ºC for 11 months decreased sharply as assessed by the level of specific antibodies generated by its inoculation in the murine model
(Lescano et al., 1998).
Toxocara species eggs are extremely resistant to many chemical disinfectant solutions and they can survive in adverse conditions, for example, 8 days in 40% formalin,
4% lysol and saturated sodium chloride, and up to 1 month in 10% formalin and hydrochloric acid (Gamboa, 2005). However, they can be destroyed by aqueous iodine, sodium hydroxide, sodium hypochlorite, temperature above 37ºC, rapid freezing to -40ºC and prolonged dryness. Since the use of sodium hydroxide remove the sticky outer protein coat, it makes the decorticated eggs more infective than eggs with an intact protein coat
(Morrondo et al., 2006).
Ethanol and sodium hypochlorite are always recommended as disinfectant agents against T. canis eggs. However ethanol is the best disinfectant because it prevents the development of the T. canis larvae in the eggs (Morrondo et al., 2006). Contrary to this, another study showed that the use of iodine solution was the best way to destroy T. canis eggs (Aycicek et al., 2001).
35 1.7 Global seroprevalence of human toxocariasis
Incidences of toxocariasis are not officially reported to the Health authorities, thus the true worldwide prevalence has not been estimated. T. canis is widely distributed in the world where dogs are infected. Multiple studies, worldwide, have demonstrated high rates (10-
87%) of soil contamination with Toxocara eggs in parks, playgrounds, sandpits, backyards and other public places. Some examples of rates of soil contamination are as follows: in
Germany (87%), Japan (42%), Norway (39%) and United Kingdom (66%) (Overgaauw,
1997; Giacometti et al., 2000). Soil contamination is a major source of human toxocariasis and led to the increase in risk of human infection.
The long-term survival of Toxocara species eggs outside the hosts (up to several years), coupled with the high reproduction of these nematodes (adult females lay about
200,000 eggs daily), is responsible for the significant contamination of soil with infective eggs. The degree of transmission of Toxocara species eggs in contaminated soil is greater in rural than urban areas. The seroprevalence studies have shown that exposure to infection in humans was higher in tropical and developing countries with low socioeconomic level than in developed countries (Overgaauw, 1997).
In tropical countries, surveys showed a positive anti-Toxocara antibody rate of
63.2% in Bali, Indonesia (Chomel et al., 1993), 84.6% in Manado, Indonesia (Hayashi et al.,
2005), 86% in Saint-Lucia, West Indies (Thompson, 1994), 92.8% in La Reunion (Magnaval et al., 1994b), 39% in Brazil (Alderete et al., 2003; Aguilar-Santos et al., 2004) and 43% in
Sri Lanka (Iddawela et al., 2003). In Malaysia, studies on various populations and ethnic groups showed a significant rate of anti-Toxocara antibody seropositivity of 11% to 58%
(Lokman Hakim et al., 1992, 1993, 1997; Patrick et al., 2001). A study by Lokman Hakim et al. (1993) showed the overall seropositivity rate was 19.6% with the highest positive rate recorded among Indians (35.5%), followed by Malays (14.8%), Chinese (10.9%) and others
(29.4%). The infection was most likely acquired from the ingestion of embryonated eggs from the soil rather than from direct contact with dogs.
36 The seroprevalence in Western countries showed that 2-5% of healthy adults from urban areas were positive compared to 14.2-37% in rural areas (Magnaval et al., 1994b). The seroprevalence rates reported were 40% in Argentina (Alonso et al., 2000), 8% in
Netherland (Lynch et al., 1988), 14% in Japan (Yoshida et al., 1999), 5% in Korea (Park et al., 2002) and 1.6% in Marche, Italy (Habluetzel et al., 2003).
The seroprevalence varies with age and among countries. All ages are at risk of getting infected but young children aged 1-7 years are at greater risk because of their play habits, their tendency to put things in their mouths and, less likely to adhere to sensible hand- washing and other hygienic habits. A study conducted at the Klang Hospital, Malaysia, revealed that the seropositivity rate was highest in children below the age of 10 (34.5%) as compared to the older age group (Lokman Hakim et al., 1993). In Ireland, Toxocara seroprevalence in schoolchildren was reported to be high (31%) and rose with age with the prevalence of ocular larva migrans (OLM) approximately 0.0121% in schoolchildren (Good et al., 2004).
The percentage of Toxocara seropositivity has been shown to be sex independent
(Figueiredo et al., 2005), but there are reports of predominant infection of T. canis in males.
The seroprevalence in Nepal was 81% with a higher percentage in males (85%) than females
(77%) (Rai et al.,1996). In general, males are more susceptible to infection than females.
This may be explained by differences in the playing and social behaviours of boys and their more active lifestyles, resulting in an increased exposure to Toxocara eggs (Overgaauw,
1997).
Many epidemiological studies have reported the association of positive Toxocara serology with allergic manifestations including asthma (Buijs et al., 1994, 1997; Lokman
Hakim et al., 1997; Patrick et al., 2001; Tonelli, 2005). A study among Malaysian children also showed that children with bronchial asthma have a higher Toxocara seropositivity
(57.8%) than the non-asthmatic controls (15.4%) (Patrick et al., 2001). The cross-sectional studies among schoolchildren aged 4-6 years in Netherland concluded that allergic manifestations occurred more often in Toxocara-seropositive children. Buijs et al. (1994)
37 also found that occurance of asthma/recurrent bronchitis was significantly associated with
Toxocara seroprevalence and suggested that toxocariasis may be a contributory factor in inducing allergic asthma. The variation in human seroprevalence of toxocariasis from country to country may also suggest that there are variations in the diagnostic tools/reagents employed in detecting the infection.
1.8 Signs and symptoms of toxocariasis
Most human infections are asymptomatic with self-resolving symptoms such as light weakness, vomiting or abdominal pain. However, clinical forms may be presented; the symptoms are due to the body's allergic reaction to the presence of the Toxocara larvae and also the migration of the larvae through the organs and tissues of the body (Despommier,
2003; Vidal et al., 2003). After Toxocara eggs are ingested, it takes at least about one week for symptoms to appear. However, the symptoms may also appear after several weeks or months and may persist for a year or even longer.
The number of infective eggs/larvae ingested (larval load), the duration of the infection, the location of the larvae, the frequency of re-infection and the degree of host immune response affect the severity of the disease (Schantz, 1989). The major clinical consequences of prolonged migration and persistence of T. canis larvae in humans are visceral larva migrans (VLM), ocular larva migrans (OLM) and covert toxocariasis (CT). A number of other disease manifestations have been attributed to this parasite as well such as neurological disease.
1.8.1 Visceral larva migrans (VLM)
VLM is caused by the migration of larvae through the human internal organs and tissues especially liver, which give rise to the inflammatory reaction. This is due to severe or repeated infection and typically occurs in children one to four years of age with more severe clinical symptoms. Pica (defined as the persistent eating of non-food substances or dirt- eating) is the main reason for the infection of children with large numbers of eggs. The most
38 common form of pica, geophagia, has been observed mostly in children under 6 years of age
(Lim, 2008).
At higher infective doses, the immune response entraps larvae in organs such as liver and lungs to give rise to clinical signs soon after infection. A very high larval load increases the probability of concurrent VLM and OLM. The classic VLM syndrome consists of episodes of fever, markedly persistent eosinophilia (an absolute eosinophil count (AEC)>
500/mm3) (Schantz, 1989); leukocytosis, hepatomegaly/splenomegaly, malaise, anemia, weight loss, coughing, wheezing, asthenia and abdominal symptoms (Rayes et al., 2001).
Progression to pulmonary and myocardial manifestations such as eosinophilic pneumonia and myocarditis has been reported (Haralambidou et al., 2005). There were also some reports of central nervous system involvement, extending to the brain (Strupp et al.,
1999, Moreira-Silva et al., 2004; Eberhardt et al., 2005). In humans, the frequency and localization of T. canis larvae in the brain is largely unknown. Of the few cases reported, larvae have been recorded in the leptomeninges and spinal cord (Dent et al., 1956), grey and white matter of the cerebrum and cerebellum (Hill et al., 1985) and the thalamus
(Beautyman et al., 1966).
Recently, Inan et al. (2006) has reported a patient who had Toxocaral VLM mimicking acute appendicitis, which led to an unnecessary operation to be performed. Renal involvement with T. canis infection is very unusual, however a case of nephrotic syndrome
(NS) in a 7 year old boy associated with T. canis infection has been reported and it was suggested that NS may be another manifestation of T. canis infection (Shetty & Aviles,
1999).
1.8.2 Ocular larva migrans (OLM)
OLM is the result of the migration of as few as a single larva to the eyes and the infection is usually unilateral. However, bilateral infections have also been reported in up to 3% of patients. Although a single larva can produce OLM, multiple larvae per eye have been reported in mice (Fenoy et al., 2001). It was suggested that if the patient had been infected
39 with a great number of eggs, the induced inflammatory and/or immunological response would have prevented any larvae from getting to the eye. This is possibly because the larvae would become encapsulated in a fibrotic granuloma. However, the combination of both presentations (VLM and OLM) may occur if the patient ingests a large number of eggs
(Kayes, 2006). OLM usually occurs in children older than 4 years old or young adults without any other clinical signs and is not strongly associated with contact with puppies. It has been suggested that ocular infection in humans occur in the early stages of infection and may be a consequence of slow larval migration following a prior infection at another site.
Symptoms may present late in the course of the disease because ocular toxocariasis is usually painless and unilateral.
The possible routes for invasion of the eye are through blood vessels, optic nerve and through the soft tissues and cerebrospinal fluid (Fenoy et al., 2001; Hayashi et al.,
2003). For entry via the cornea or anterior sclera, the larvae would have to be deposited on the front of the eye but this is unlikely to happen (Taylor et al., 2006). Clinical presentation varies according to the anatomic site of involvement: peripheral retina and vitreous, posterior pole endophthalmitis, optic nerve and anterior segment. However, involvement of the peripheral retina and vitreous either separately or both are the most common (Kayes, 2006).
Ocular toxocariasis may cause decreased visual acuity, chorioretinitis, uveitis, keratitis, leukocorea (white appearance of the pupil), strabismus, retinal detachment and sudden loss of vision (Stewart et al., 2005). Unilateral loss of vision is believed to be the major cause of unilateral blindness in children of school age. The unilateral loss of vision in these children may remain unnoticed unless a strabismus develops and/or a leukocorea is apparent. Gillespie et al. (1993) reported the findings in 33 patients with OLM and all with positive Toxocara serology with symptoms of visual loss (26 patients), eye pain (8 patients), retinal abnormality (17 patients), uveitis (20 patients) and endophthalmitis (9 patients). It is rarely accompanied by eosinophilia or a raised immunoglobulin E (IgE) concentration.
Serum antibodies to TES products are few or absent and may be evident only in vitreous specimens. Humans are fortunate because they are less susceptible to ocular infections
40 compared to animals (Taylor, 2001). However, Good et al. (2004) reported a statistical association between ocular toxocariasis and convulsion in humans.
1.8.3 Covert toxocariasis (CT)
In the covert form, antibodies to Toxocara are associated with a few systemic or localized symptoms that do not correspond to the other two syndromes (VLM and OLM). The symptoms may include abdominal pain, cough, headaches, sleeping difficulty, behavioral problems, wheezing, hepatomegaly and lymphadenitis and the symptoms can last for months or years. In France and Ireland, patients who had non-specific symptoms including fatigue, abdominal pain, nausea, fever, lymphadenopathy, with or without moderate eosinophilia showed positive anti-Toxocara antibody test (Schantz, 1989).
T. canis has also been implicated as a possible cause of idiopathic seizure disorders.
The covert form is not always associated with eosinophilia. Covert toxocariasis has been associated with asthma (Buijs et al., 1994), functional intestinal disorders and skin disorders
(Humbert et al., 2000).
1.8.4 Neurological toxocariasis
Though neurological manifestations of toxocariasis are rare, it remains an important differential diagnosis of various neurological disorders (Eberhardt et al., 2005; Finsterer &
Auer, 2007). As VLM, the clinical signs of neurological toxocariasis are non-specific, leading to possible under diagnosis of this condition. The migration of T. canis larvae through the tissue of central nervous system results in varying degrees of pathology.
Manifestations of central nervous system infection are dementia, eosinophilic meningo- encephalitis (EME), myelitis, cerebral vasculitis, epilepsy or optic neuritis. In addition, the association between Toxocara seropositivity and increased risk of epilepsy has also been reported. Children who have a history of epilepsy showed a statistically significant increase in antibody against Toxocara antigens (Nicoletti et al., 2002). Manifestations of peripheral nervous system infection are radiculitis, affliction of cranial nerves or musculo-skeletal
41 involvement. Early recognition and treatment of the infection are important since it reduces morbidity and mortality and the risk of secondary superinfection (Magnaval et al., 2001).
1. 9 Pathogenesis of human toxocariasis
The pathology may be due to immune responses either to larvae themselves or to their products released in the tissues of the host. Tissue damage is due to the host inflammatory reaction more than the infection itself. The inflammation resulted from the pathologic manifestation is primarily caused by the immune response directed against the excretory- secretory antigens of the larvae. The antigens are released from their outer epicuticle coat, which is readily sloughed off when bound to specific antibodies or under attack by host inflammatory cells. Toxocara larvae tissue invasion or death of larvae induced delayed-type hypersensitivity responses in human hosts. An acute inflammation consists of aggregation of inflammatory cells including eosinophils, neutrophils and monocytes (Shetty & Aviles,
1999).
The most common and immediate host response is a reactive eosinophilia and the synthesis of IgE antibodies. These antigens induce a host immune response characterized as a T helper 2-type (Th-2) CD4+ cellular immune response characterized by the production of interleukin 4 (IL-4) that promotes the switching of B-cell isotypes to the production of IgE and IL-5. These, in turn, promote eosinophil differentiation and vascular adhesion. These host reactions are the major drivers for the resulting immunopathologic processes observed in these patients (Shetty & Aviles, 1999).
Development of a hypereosinophilia syndrome may be the result of uncontrolled eosinophil proliferation. Allergic hypersensitive reactions (type I) may be mediated by the high levels of IgE, while the formation of IgE and anti-IgE antibodies may mediate immune complex-associated reactions of the type III hypersensitivity responses. These two types of immune reactions have been associated with the clinical course of toxocariasis (Shetty &
Aviles, 1999).
42 The mechanism by which larvae in tissues are killed and eliminated is not known. In mice, eosinophils do not appear to be involved in host resistance, since antibody-dependent cell cytolysis mediated both by eosinophils and specific IgE antibodies is lacking. Trapping of larvae in the liver during somatic migration may play a significant role in the host defense. At the point where they die, small abscesses or granulomas (small masses of inflamed tissue) may develop (Despommier, 2003).
Most of the larvae in the tissues are not associated with inflammatory cells. This is due to the rapid migratory rate of the larvae through the tissues. The inflammatory reaction causes epitheloid cells to surround each larva. By one month, the larvae are surrounded by collagen fibers, and in chronic infections, most of the larvae are sheathed by mature granulomas. A few larvae in the granulomas are unscathed, and presumebly to be still metabolically active (Lampariello & Primo, 1999).
At low infective doses, the antigenic mass is insufficient to stimulate a protective immune response, to cause larval death, and larvae can survive longer. These patients probably remain asymptomatic and do not even know if they have had infection unless a larva causes ocular damage. However, at higher infective doses, the immune response entraps larvae in organs such as the liver and lungs giving rise to clinical signs and symptoms relatively soon after infection (Glickman & Schantz, 1981).
1.10 Immunological aspect of toxocariasis
1.10.1 Cellular immune responses
Infection with T. canis larvae induces adaptive immunity in their hosts, which are characterized by high and persisting eosinophilia, tissue mastocytosis, production of cytokines typical for a Th-2-type immune response (IL-4, IL-5 and IL-10) and increased production of IgE antibody. The associations between eosinophilia and infection by T. canis have also been reported (Alonso et al., 2000; Chiodo et al., 2006). Buijs et al. (1997) have reported that total serum IgE levels and blood eosinophils were significantly higher in the
Toxocara-seropositive patients than in the seronegative groups. A result of this type of
43 immune response is the establishment of long-lasting parasitization of larvae in the muscles and brain (arrested stage) with only partial elimination of larvae (Kayes, 2006).
1.10.2 Antibody subclasses in toxocariasis
1.10.2.1 IgM
In the humoral response to an antigen, specific IgM is the first subclass produced and levels are detectable as early as 5-7 days after antigen encounter. IgM titres usually decline to background levels as IgG titres rise but in canine toxocariasis high IgM levels persist for months. In naturally infected puppies, IgM levels increase steadily for the first three months of life and persist until adulthood eventhough there is no correlation between IgM titres and adult worm burdens (Arango, 1998).
Some investigators reported that the diagnosis of acute Toxocara infection is best made by the demonstration of raised Toxocara-specific IgM (Shetty & Aviles, 1999).
Besides IgG1 and IgE, level of IgM antibody was shown to be elevated in toxocariasis patients (Reiterova et al., 2003). However, only a few studies reported the usefulness of specific IgM for serodiagnosis of human toxocariasis. A case report of VLM showed elevated Toxocara specific IgG and IgM titres, and following treatment the IgM titres decreased but IgG titres were elevated (Arango, 1998).
1.10.2.2 IgE
High total IgE is a hallmark of visceral infections by parasites and total IgE level is well correlated with the presence of larva. High levels of non-specific IgE antibodies have been described in several experimental and natural nematode infections. For example, total serum
IgE levels were significantly increased in patients with toxocariasis however Toxocara- specific IgE was not detected or was at a very low level (Uhlikova et al., 1996).
High level of IgE specific for TES has also been reported in the serum of patients with clinical signs suggestive of Toxocara infection. Sera containing greater than 500
Toxocara units/l of IgE are classified as toxocariasis positive cases (Magnaval et al., 2001).
44 Figuieredo et al. (2005) have observed extremely significant associations between positive serology for T. canis with elevated IgE levels (above 200 IU/dl) and eosinophilia (above
400 cells/mm3). Specific IgE occurred significantly more often in the Toxocara seropositive group than in seronegative group. VLM patients had been reported to have 10-15 times the average normal level of IgE as determined by the radioimmunosorbent test (Hogarth-Scott et al., 1969).
Levels of antigen-specific IgE antibodies correlated with the degree of protection.
The role of IgE in protective immunity was also demonstrated in other nematode infections.
A study of the time course of production of nematode-specific versus non-specific IgE showed that, after re-infection, most of the total IgE response was associated with the parasite-specific IgE, implying that parasite-specific B and T cells on re-stimulation with worm antigens produced factors which stimulated parasite-specific responses (Moreira-Silva et al., 2002).
A previous study showed that the reactivity of IgE against SDS-PAGE separated
TES antigens was higher in toxocariasis patients (35%) than in asymptomatic patients
(24%). Significantly elevated levels of IgE/anti-IgE immune complexes were detected in sera of patients with VLM and OLM. IgE or anti-IgE immune complexes might possibly participate in VLM and OLM by inducing type III hypersensitivity (Obwaller et al., 1998).
In OLM, serum IgE titer is lower than in VLM because the worm burden is lesser.
Brunello et al. (1983) reported good correlation between IgG and IgE in detecting larva-specific antibodies. In the VLM patients, IgG antibodies seem to mask the presence of
IgE by a competitive mechanism toward the same epitopes of the parasite. In fact, IgE antibody titers increased after depletion of IgG from serum. Elefant et al. (2006) have suggested that IgE antibodies plus eosinophil counts are useful parameters for patient follow- up after chemotherapy. Toxocara specific IgE assay has also been reported to be helpful in reducing the problem of cross-reactivity in immunoassays (Wiechinger, 1998; Magnaval et al., 2001).
45 1.10.2.3 IgG subclasses
The antigen-specific IgG responses appear from the second week of infection (Bowman et al., 1987). Anti-Toxocara IgG antibody is usually the subclass targeted in the serodiagnostic tests because of its abundance in serum and its increased avidity and affinity, due to the anamnestic response (Smith & Rahmah, 2006). The IgG1 subclass is a dominant antibody in toxocariasis and a good indicator of infection. However other IgG subclasses may also be useful in monitoring disease progression and post-treatment, as well as in the diagnosis of human toxocariasis (Magnaval et al., 2001). Many studies have reported the potential value of specific IgG subclass antibodies in diagnosing various helminthic infections including toxocariasis, gnathostomiasis, filariasis, echinococcosis, neurocysticercosis and sparganosis
(Grimm et al., 1998; Yang et al., 1998; Chung et al., 2000; Rahmah et al., 2001). The detection of IgG subclasses other than total IgG can be helpful in the differential diagnosis of human toxocariasis. The serum ELISA for Toxocara-specific IgG is less sensitive for the diagnosis of OLM than for other forms of the disease.
Higher IgG1 levels were detected in serum of mice infected with T. canis than in uninfected controls (Cuellar et al., 2001; Fan et al., 2004). A study performed by Obwaller et al. (1998) showed that TES-specific IgG1 subclass reactivity by TES ELISA was similar in both symptomatic (VLM, OLM) and asymptomatic persons. Analysis of IgG subclasses specificity against TES antigens indicated that IgG1 was the predominant immunoglobulin followed by IgG2, IgG4 and IgG3 in all three groups of patients. Within the T. canis- seropositive patients, IgG1, IgG2 and IgG4 levels were significantly higher (P<0.001) in the
VLM patients than in the asymptomatic persons. However, only IgG1 titres were significantly higher in OLM patients compared with asymptomatic persons.
The IgG1 subclass is considered in mice to be the second class of anaphylactic antibodies (eosinophil-dependent), able to induce histamine release from the mast cells. On the other hand, no significant difference in titres of IgG3 and IgG2 antibodies, indicators of
Th1-type responses, was seen between uninfected and infected groups of mice (Fan et al.,
2004). Watthankulpanich et al. (2008) reported that IgG2 gave the greatest sensitivity (98%)
46 followed by IgG3, IgG4, IgG1 and IgG while IgG3 gave the greatest specificity (81%) followed by IgG1, IgG, IgG2 and IgG4.
Detection of Toxocara-specific IgG4 has been shown to greatly increase the specificity of Toxocara serodiagnosis and significantly reduce the cross-reactions with antibodies to other helminthic infections, but less sensitive than total IgG TES-ELISA
(Wiechinger, 1998; Rahmah et al., 2005, Norhaida et al., 2008). Previous study indicated that IgG4-based immunodiagnosis for toxocariasis produced less false positive results than
IgG-based immunoassay when tested with serum samples from other parasitic infections. A study done by Rahmah et al. (2005) showed that the sensitivity of IgG TES ELISA and IgG4
TES ELISA was 97.1% and 45.7% respectively, while the specificity was 36.0% and 78.6%, respectively. Another study by Norhaida et al. (2008) showed that rTES-30USM IgG4-
ELISA gave better specificity compared to IgG-ELISA. This suggested that an IgG4 immunoassay may be useful for the secondary screening of recombinant clones in an effort to develop improved serological tests for toxocariasis (Rahmah et al., 2005).
1.11 Diagnosis of human toxocariasis
1.11.1 Clinical diagnosis
A definitive diagnosis of toxocariasis is always a challenge to clinicians, since the symptoms are non-specific and mimic other helminthic parasite infections, allergies and asthma. T. canis infection can be suspected in at risk children, such as those with puppies at home, have had contact with soil and showed symptoms of hepatomegaly and/or asthma with eosinophilia and increased serum IgE (Figueiredo et al., 2005). Clinical diagnosis include a selection of symptoms in each of the disease type, i.e. VLM, OLM, covert toxocariasis and other non-specific forms such as pulmonary, cutaneous and neurological manifestations.
Toxocariasis can be suspected when eosinophilia occurs, but this sign can also be associated with other helminthic infections and other diseases. Thus, the diagnosis of toxocariasis is not usually solely based on clinical signs and symptoms. A definitive laboratory diagnosis of human Toxocaral infection can be achieved by histopathology
47 examination of various organ specimens, such as liver, brain, lung and enucleated eye.
However, identification of L2 larvae in biopsy tissue samples is not always successful because there are often a few parasites in the affected tissues.
1.11.1.1 Radiology
Imaging studies in toxocariasis depend on the location of the disease. Medical imaging techniques can be used to detect and localize granulomatous lesions due to migrating
Toxocara larvae in tissues and to support a tentative diagnosis of toxocariasis. Abdominal ultrasonography is a good screening test to identify granulomas in the liver and has shown multiple hypoechoic areas in the livers of children who initially presented with hepatomegaly, eosinophilia and a positive Toxocara serology (Baldisserott et al., 1999;
Chang et al., 2006).
Echocardiography is used to evaluate myocardial function, which is depressed in myocarditis, and to identify pericardial fluid and intracardiac pseudotumors. Chest radiography is indicated in patients with wheezing and eosinophilia in whom eosinophilic pneumonia is suspected. It also can be used to identify pleural effusions and cardiomegaly.
Computed tomography scanning and magnetic resonance imaging (MRI) have also been used, particularly for evaluating central nervous system diseases.
1.11.2 Laboratory diagnosis
1.11.2.1 Parasitology tests
The eggs are not found in the human stool because the larvae of T. canis do not mature in humans. Repeated stool examinations and Baermann’s method for detection of
Strongyloides stercoralis larvae, should be performed to rule out other parasitic infections.
The negative stool examinations in the presence of eosinophilia and elevated serum IgE levels are indications for specific immunodiagnostic tests for parasitic infections including anisakiasis, ascariasis, strongyloidiasis, trichinellosis and toxocariasis (Smith & Rahmah,
2006).
48 1.11.2.2 Serology tests
The diagnosis of toxocariasis is often difficult in humans, leading to a high index of suspicion and must be supported by detection of significant concentrations of anti-Toxocara antibodies in the serum. As direct detection of larvae is not feasible in humans the clinically suspected cases are usually confirmed by serological methods (Magnaval et al., 2001).
A few non-defined antigens have been used for the serodiagnosis of human toxocariasis prior to the use of TES, such as soluble somatic L2 antigen, soluble embryonated egg extract and soluble adult antigens. However, many of these somatic antigens lacked specificity and were cross-reactive especially with sera from patients infected with Ascaris lumbricoides and other ascaridids (Smith, 1998).
The serological assay using native TES antigen, is presently the most sensitive method for diagnosis of human toxocariasis. The use of larval antigens offers greater sensitivity to serodiagnosis, and specificity can be enhanced by absorbing the sera with
Ascaris antigens (de Savigny, 1975). Currently, TES is the most commonly used antigens in antibody detection assays such as indirect antibody competition ELISA (IACE), radio- iodination, Western blot, latex agglutination, radio-allergosorbent test (RAST), direct and indirect fluorescent antibody tests (IFAT, DFAT), haemagglutination test, in vitro L2 fluorescence, sandwich ELISA, counter immunoelectrophoresis (CIEP), double gel diffusion test (DGDT), paper radio-immunosorbent test (PRIST), rapid test (ToxocaraCHEK), indirect
TES ELISA and dot-ELISA. Most of these assays are based on IgG, others used IgM and
IgE antibodies. However, the indirect IgG TES-ELISA is currently the most common test employed for serological diagnosis of human toxocariasis, in laboratory-based assays and in commercial kits (Smith & Rahmah, 2006).
A simple ELISA using TES material has been introduced by de Savigny (1975) but it has also been shown to be cross-reactive with other nematode infections (Page et al.,
1991). The use of TES antigens from culture of the second stage of T. canis larvae, maintained in vitro further significantly increases the sensitivity of the ELISA. ELISA has
49 been shown to be as sensitive as RIA with TES antigens and exhibits a high degree of specificity (Jacquier et al., 1991).
A previous study has been conducted to evaluate the dot-ELISA as a diagnostic test
(Roldan et al., 2006). Dot-ELISA for the detection of IgG antibodies has been reported to have a comparable sensitivity with standard ELISA (100%) and exhibited better specificity
(95%) since only three cross-reactions occurred in the dot-ELISA, compared with six in the standard ELISA and large-scale evaluation has not been performed. However, these assays that are based on TES may have similar problems when used in the tropical countries where polyparasitism are endemic.
Several commercial Toxocara enzyme immunoassay kits have been used for serodiagnosis, such as Toxocara Ab IgG Cypress Diagnostics (Belgium), Toxocara IgG
CELISA (Australia), Toxocara CELISA (United Kingdom), NovaTec IgG ELISA
(Germany), NOVUM Toxocara canis IgG/IgM EIA (Germany), Toxocara canis IgG (Czech
Republic), Toxocara canis ELISA kit (Switzerland) and IBL-IgG ELISA (Hamburg).
However, these TES-based assays show high specificity only if used in developed countries where helminthic infections such as soil-transmitted helminthiases (STH) are not endemic.
Many sera from patients infected with STH particularly those with Trichuris trichiura infections were positive with these kits. Although there may be co-infection between
Toxocara and STH, the degree of cross-reaction observed was too high to be solely due to this possibility (Nunes et al., 1999).
Cross-reactivity is a major problem with Toxocara serodiagnosis in tropical countries especially with antibodies to STH infections such as A. lumbricoides, T. trichiura, hookworms and S. stercoralis. Thus, TES may not be sufficiently specific for use in countries where STH infections are prevalent (Lynch et al., 1988; Rahmah et al., 2005). The commercially available ELISAs may only be useful for differential diagnosis, i.e. when the results are negative and the diagnosis of toxocariasis can be excluded, thus avoiding unnecessary treatment. However, interpretation of the result may be problematic when the test results are positive (Rahmah et al., 2005).
50 Many studies have reported the potential value of specific IgG subclass antibodies in diagnosing toxocariasis. The detection of immunoglobulin subclasses other than IgG1, such as the use of anti-Toxocara IgG4 in ELISA has been shown to increase the specificity of the serological diagnosis of toxocariasis (Wiechinger, 1998; Rahmah et al., 2005, Norhaida et al., 2008), and therefore, can be helpful in the differential diagnosis of toxocariasis. This situation is similar in the cases of lymphatic and non-lymphatic filariasis, whereby the use of anti-IgG4 antibody greatly increases the specificity of the serological assays and is accepted as a marker of active infection (Kwan-Lim et al., 1990; Weil et al., 1990; Lal & Ottesen,
1998; Rahmah et al., 2001; Klion et al., 2003).
The use of IgG4 and IgE antibodies in TES ELISA have also been shown to reduce the cross-reactions from other helminthic infections, thus greatly increasing the specificity of the assay but is less sensitive than total IgG TES ELISA in diagnosing of human toxocariasis
(Wiechinger, 1998; Rahmah et al., 2005; Norhaida et al., 2008). Norhaida et al. (2008) reported that the assay based on IgG4-rTES-30USM demonstrated a sensitivity of 92.3% and a specificity of 89.6%, while the commercial kit (IgG-TES) exhibited a sensitivity of 100% and a specificity of only 55.7%. Furthermore, no detection test based on IgG4 is yet available commercially for the detection of Toxocara infection.
Another diagnostic format has been developed using polysiloxane/polyvinyl alcohol beads coupled with recombinant T. canis antigen for the immunodiagnosis of human toxocariasis and its usefulness has been demonstrated (Coelho et al., 2003). However, no further evaluation was performed.
1.11.2.2.1 IgG antibody avidity
In general, it is considered that blood eosinophilia combined with positive serological tests indicate active toxocariasis. However, antibody levels cannot be used to discriminate between recent and chronic infections. Determination of specific IgG antibody avidity has
51 been shown to be useful in the diagnosis of various parasitic diseases including toxoplasmosis, neosporosis, fascioliasis and Trypanosoma cruzi infections in order to distinguish between reactivation of 'dormant larvae', re-infection or primary infection, especially in situations when differentiation of recent and distant infections is crucial (Abou-
Basha et al., 2000; Hubner et al., 2001).
Thus, recently acquired Toxocara infection can be distinguished from chronic/past infection by assessing the avidity of the IgG antibodies (Hubner et al., 2001; Dziemian et al.,
2008). It is known that the avidity of specific antibodies increases with time after antigen challenge and assessment of the degree of avidity can be used to discriminate between recent and distant infections. An IgG avidity assay for the diagnosis of acute toxocariasis has been developed (Hubner et al., 2001; Dziemian et al., 2008).
The avidity index was calculated as the ratio of IgG values in sera treated with urea and the value of IgG in non-treated sera, multiplied by 100. An index less than 40 was considered low avidity indicative of recently acquired infection, 36-40 was borderlined and more than 40 was considered high avidity indicative of past infection. There were only
5.09% of sera from 1376 patients with low avidity antibody, indicating recently acquired infection. Thus, determination of antibody avidity, together with IgG, IgE and IgG subclass reactivity can help to improve Toxocara serodiagnosis (Hubner et al., 2001).
Another study performed on 150 sera from children with visceral toxocariasis and in
46 sera from children with ocular toxocariasis showed that 94·2% of positive sera collected from patients reporting infection more than 6 months ago had high IgG avidity values, confirming distant toxocariasis, whereas 25·9% of positive sera taken less than 6 months after infection showed low indices of IgG avidity. The results suggested that measurement of the specific IgG avidity may assist in discriminating between recent and distant toxocariasis. The method can be used effectively to rule out (due to high avidity) a recently acquired infection. Low avidity is less reliable in discriminating between recent and distant infections (Dziemian et al., 2008).
52 1.11.2.2.2 Western blot (WB)
Another method that can be used to detect anti-Toxocara antibodies is by Western blot analysis. Immunoblot technique has been used for the identification of the immunodominant antigenic fractions and also to confirm the specificity of the antibodies from sera of patients with proven diagnosis of toxocariasis (Magnaval et al., 1991; 2002). TES WB assay was able to identify potential antigens for serodiagnosis and their discrimination from other cross-reactive antigens in TES. The cross-reactivity to various helminthic infections was seen among high molecular weight of 132, 147 and 200 kDa banding patterns (Park et al.,
2002). Low molecular weight antigens (24, 28, 30 and 35 kDa) were more specific to the genus Toxocara and less affected for cross-reactivity (Magnaval et al., 1991). Analysis of some ascaridid cross-reactions by immunoblot revealed that antigens > 50 kDa were cross- reactive. Lynch et al. (1988) reported that 81 kDa antigen, while Nunes et al. (1999) identified a ~ 55-66 kDa cross-reactive antigen complex was responsible for the strong cross-reactivity between T. canis and Ascaris extracts.
Preabsorption of patients' sera with A. lumbricoides antigens have been proven to reduce cross-reactions (Lescano et al., 1998; Anurama Filho et al., 2003). A previous study which was performed by Nunes et al. (1999) demonstrated that a band with a molecular weight around 55-66 kDa that was responsible for the cross-reactivity between T. canis and
A. suum disappeared when absorption of serum samples with A. suum was performed.
A commercially available IgG TES WB kit is Toxocara Western Blot IgG (Testline,
Czech Republic). A series of transverse bands are apparent on WB strips corresponding to positive reaction of antibodies with individually separated antigens of respective molecular masses. Antigens with molecular masses of 120 triplets, 70, 55, 40, 32 and 26 kDa are characteristic for Toxocara. Antigenic bands of molecular weights 120 kDa, 32 kDa and 26 kDa are considered as highly specific and findings of all these bands are evaluated as positive. Finding of at least one highly specific or any other characterizing band is evaluated as negative. Finding of any other band combination is evaluated as borderline.
53 1.11.2.3 Rapid test
To date, there is only one commercially available rapid test for diagnosis of human toxocariasis. A flow-through rapid screening kit, ToxocaraCHEK was developed by Akao et al. (1997) for detection of anti-Toxocara larval TES antibodies in human serum and can be performed within three minutes. The principle of the test is the immobilization of captured larval TES antigen (0.5 µg/device) on the nitrocellulose membrane of the test device. Anti-
Toxocara antibodies in the human sample are trapped by the immobilized antigen. The presence of bound antibodies is revealed by subsequent treatment with a protein A-colloidal gold conjugate which binds to form a red color on the membrane.
This rapid kit was reported to show high correlation with ELISA (ELISA NOVUM and ELISA PU), immunoblot and double gel diffusion tests (DGDT) but it has lower specificity compared to ELISA (Akao et al., 1997; Dubinsky et al., 2000). Out of 14 different human parasitoses diagnosed for the presence of anti-Toxocara IgM and IgG antibodies by this test, cross-reaction was observed only in gnathostomiasis sample. This rapid kit is thought to be suitable as a screening test (Dubinsky et al., 2000).
1.11.2.4 Other assays
Other assays that have been used to confirm clinical diagnoses are complement fixation test
(CFT), intradermal sensitivity, bentonite flocculation, larval precipitation and capillary tube precipitation (Mirdha & Khokar, 2002).
A few other tools can be used for definitive diagnosis of toxocariasis through the specific detection of Toxocara DNA in tissue from infected hosts such as PCR (Rai et al.,
1997; Wu et al., 1997), RFLP, RAPD, sequencing of Toxocara nuclear ribosomal (rDNA), mitochondrial DNA (mtDNA) or repetitive DNA (Zhu et al., 2001), but these are rarely done due to difficulty in acquiring biopsy samples. PCR has been used to detect DNA in experimental murine liver biopsy material but the use of PCR for detecting Toxocara DNA in human biopsy sample has not been reported so far. However, some of the methods such as
54 PCR and RAPD have some limitations, including reproducibility problem and non-specific amplification due to the nature of the primers used (Rai et al., 1997).
1.11.3 Diagnosis of OLM
1.11.3.1 Clinical diagnosis
The definite diagnosis of ocular toxocariasis is difficult to establish since the larvae are rarely identified from the lesions. The clinical features of systemic or VLM are usually absent when there is ocular involvement, probably due to the low larval burden (often a single larva). At present, the diagnosis of ocular toxocariasis is usually presumptive and based on clinical findings, especially ophthalmoscopic appearance. However, it is difficult to establish the diagnosis of ocular toxocariasis based on clinical manifestations solely, because ocular symptoms may be diverse and inflammatory signs such as redness and pain are not always present. Other species of larvae migrans and intraocular parasites may mimic the reactions observed in ocular toxocariasis. Non-localizing reactions such as anterior uveitis, vitritis or diffuse choroiditis may be very difficult to differentiate from other more common causes of these manifestations. Sometimes, a mobile larva can be directly observed under the retina (Despommier, 2003).
Other methods that can be used are ultrasound biomicroscopy (USB), computerized tomography scan, ultrasonography and MRI studies which can reveal a highly reflective, peripheral mass, vitreous bands or membranes and traction retinal detachment (Tran et al.,
1999; Fenoy et al., 2001; Sabrosa & de Souza, 2001).
1.11.3.2 Laboratory diagnosis
The major differential diagnosis of ocular toxocariasis in humans is retinoblastoma, which was the reason for enucleation of eye because of the fear of involvement of the other eye.
The use of TES antigen can prove useful compared to T. canis embryonated egg antigen since TES antigen can clearly differentiate between sera from OLM and retinoblastoma cases.
55 The serodiagnosis of ocular toxocariasis is difficult since serum anti-Toxocara antibody levels are usually low or undetectable either by IgG TES-ELISA or by the more sensitive Western blot (Magnaval et al., 2002). Furthermore, eosinophilia is often absent in
OLM cases. However, detection of specific antibodies in aqueous/vitreous fluids by IgG
TES-ELISA is considered as the most important feature and more reliable than that in serum for confirmation of OLM cases, since IgG antibody titers in vitreous fluids are much greater than in the serum. Anti-Toxocara antibodies when found in aqueous/vitreous fluids of patients with clinical signs of OLM, should be considered diagnostic for OLM. The higher titre of anti-TES antibody in ocular fluids than in serum is suggestive of local antibody production (Visser et al., 2007). This was confirmed whereby TES ELISA using patient's serum was negative in a case of OLM with an optic disc granuloma. A recent study has reported cases in which the aqueous humour antibody titre was positive although the serology titre was negative (Magnaval et al., 2002). This shows that measuring anti-
Toxocara antibody titres in intraocular fluid may be helpful in diagnosing ocular toxocariasis.
Five cases of ocular toxocariasis confirmed by ELISA with specific anti-Toxocara
IgE antibodies and immunoblot that uses specific IgG antibodies have been reported (Park et al., 2002). Recent study by Visser et al. (2007) showed that serological screening is not informative for the diagnosis of intraocular Toxocara infection. Toxocara Goldmann-Witmer coefficient (GWC) analysis might be of value when diagnosing patients with posterior focal lesions or vitritis of unknown etiology. This analysis was determined by the following equation: [specific IgG in aqueous humors/specific IgG in serum]/ [total IgG in aqueous humors/total IgG in serum]. Intraocular antibody production was considered positive when the GWC value exceeded three. However, T. canis-specific serum and aqueous humor IgG were determined by T. canis IgG-ELISA kit which uses native TES antigen and thus not highly specific.
56 Other methods reported for differential diagnosis between OLM and retinoblastoma was gel precipitation (electrosyneresis), Ouchterlony double diffusion and immunoelectrophoresis (Park et al., 2002).
1.12 Recombinant TES antigens in serodiagnosis
Since the production of TES is limited to the capacity of parasite culture, the use of recombinant antigens which can be produced in unlimited amounts, offers a good alternative for diagnosis of human toxocariasis. Several researchers have reported recombinant antigens that are potentially useful for serodiagnosis of toxocariasis. Recombinant antigens should offer further solutions for Toxocara serodiagnosis by providing increased sensitivity and specificity, compared with native TES. The recombinant antigens must share important antigenic and immunogenic structures with native TES. Important candidates reported thus far are rTES-30 and rTES-120 recombinant antigens, which are of potential value for the serodiagnosis of human toxocariasis.
1.12.1 rTES-30
Yamasaki et al. (1998) have developed a recombinant T. canis second-stage larva antigen corresponding to the 30-kDa protein of the TES secreted by infective larvae and evaluated its usefulness by comparing its specificity with that of TES using a total of 153 serum samples from patients with 20 different helminthic infections. The rTES-30 recombinant antigen in
IgG assay was demonstrated to be sensitive and specific for detection of human toxocariasis.
However, the assay requires further evaluation since there was some cross-reactivity, especially in Western blots (Yamasaki et al., 1998; 2000; Coelho et al., 2005). Western blot analysis showed that rTES-30 reacted specifically with sera from toxocariasis patients, but not with sera from B. malayi infections, ascariasis and dirofilariasis. Cross-reactions with anisakiasis sera were diminished when these sera were pre-absorbed with Anisakis antigen
(Yamasaki et al., 1998).
57 This recombinant antigen (namely rTES-30USM) previously genetically engineered by assembly PCR and was shown to be highly sensitive and specific in IgG4-ELISA compared to IgG-ELISA (Norhaida et al., 2008). Thus, this recombinant antigen seemed to be highly specific for toxocariasis and thus may be a reliable diagnostic marker.
1.12.2 rTES-120
T. canis expresses a family of mucins, which are closely associated with the surface coat or glycocalyx of the larval parasite and migrates around 120 kDa on SDS-PAGE (Gems &
Maizels, 1996). The mobility on SDS-PAGE is not the same with their actual mass values of
40-45 kDa because of extensive glycosylation. TES-120 produced four discrete bands on
SDS-PAGE with an estimated molecular weight of 120 kDa (Loukas et al., 2000a). The mRNA for TES-120 is hyperabundant, representing approximately 10% of the complementary DNA. It was suggested that the TES-120 components are secreted physiologically through the buccal cavity and the secretory pore. TES-120 is the most abundantly synthesized larval protein, rich in serine and threonine (the acceptor residues for
O-glycosylation).
To date, five distinct mucins have been isolated (Tc-muc-1, Tc-muc-2, Tc-muc-3, Tc- muc-4 and Tc-muc-5). Analysis on native TES products using monoclonal antibody showed that Tc-muc-1 is the most prominent while Tc-muc-4 is expressed at a relatively low level
(Tetteh et al., 1999). Tc-muc-5 has a larger open reading frame and a more divergent sequence than other T. canis mucins, suggesting that this protein is not part of TES-120 family of surface coat proteins (Doeden et al., 2001). These proteins are expressed and secreted at a high level, indicating a primary role in immune evasion by parasites (Loukas et al., 2000a).
Tc-muc-1 gene which encodes rTES-120 recombinant antigen produced in the bacterium E. coli and methylotropic yeast expression systems has been shown to react only with sera from toxocariasis patients. Thus, this highly specific rTES-120 recombinant antigen was reported to be of potential diagnostic value for detection of human toxocariasis
(Fong et al., 2003; 2004) but it has not been well-tested.
58 1.13 Treatment, management and prognosis
Most toxocariasis patients recover without treatment. Majority of them will have no symptoms at all, never diagnosed and will never realise that they have had the infection.
Whether toxocariasis patients need to be treated, depends on the type and severity of clinical signs. However, as human toxocariasis is a chronic infection, which often lasts for several years and re-activation of circulation of dormant larvae may occur, effective treatment is the choice for control of human disease. Specific parasiticidal agents such as diethylcarbamazine
(DEC) or thiabendazole have been successfully used for treatment. Therapy with DEC is accepted as one of the most effective. However, this drug can elicit severe allergic reactions in patients. Corticosteroid treatments have been used with and without specific anti-larval therapy with some reports of improvement in severe cases like myocarditis (Schantz, 1989).
Treatment of ocular toxocariasis is more difficult and usually involves steroid to prevent or reduce inflammatory reactions to the worm in the eye and to prevent progressive damage to the eye. This may include laser photocoagulation to kill the worm before it reaches the macula in addition to drug therapy. Surgery has been used in conjunction with vitrectomy to remove the retinal parts of the granuloma and also where traction bands have caused retinal detachments (Stewart et al., 2005).
1.14 Statement of the problem and rationale of the study
One of the difficulties in diagnosing toxocariasis is the lack of pathognomonic signs and symptoms. As the larvae remain in internal organs, the definitive laboratory diagnosis is by histopathological examination of tissue specimen but this is rarely done. To date, the laboratory diagnosis of human toxocariasis is by serology, since the parasite does not produce any stage that can be detected in stool. Accurate diagnosis of toxocariasis will also allow accurate information on the epidemiology of toxocariasis in the country. This in turn can lead to better preventive measures, which will contribute to improved well-being of the society.
59 Currently, the most common routine serology test used is an indirect commercial
IgG-ELISA kit, which employs TES antigen obtained from in vitro culture of T. canis second-stage larvae. However, these tests lack specificity. The specificity issue may not be a major concern in temperate developed countries where parasite co-infection are uncommon and infections with STH are less prevalent, however, it is a significant problem for serodiagnosis in the tropics. Commercial IgG-ELISA kits using native TES antigen will prove useful for excluding toxocariasis from differential diagnosis, but when the results are positive, interpretation of the test can be problematic (Rahmah et al., 2005). The relatively low specificity of the current available serodiagnostic assays for usage in tropical countries where polyparasitism is endemic has called for an alternative diagnostic assay for toxocariasis. Thus, the development of specific, sensitive and reliable assay to demonstrate the presence of antibody in toxocariasis is needed towards improving serodiagnosis.
TES products have played an important role in the diagnosis of human toxocariasis.
However the use of native TES antigen from larval medium for the serodiagnosis of human toxocariasis has several limitations. The sensitivity and specificity of TES-serodiagnostic assay vary among batches because of the differences in TES preparation. The contamination of TES soluble somatic antigens from dead or degenerating larvae may occur if the dead larvae are not regularly removed from cultures by Baermannization. The heterogeneous composition of TES with its wide range of protein masses may increase the possibility of cross-reaction with antibodies produced to other helminthic infections. The dead larvae in the culture medium may 'contaminate' the TES preparation. This may lead to the cross- reactions especially with STH sera. Besides that, infected puppies have to be sacrificed as a continuous source of larvae. It is time-consuming and the culture medium needs to be routinely changed in order to ensure survival of the larvae and decrease variability in quality and purity (Smith & Rahmah, 2006). Thus, an alternative source of TES antigens is highly desirable.
The use of recombinant antigen offers significant benefits for future diagnosis since the capacity of parasite culture limits the production of TES (Yamasaki et al., 2000). The
60 recombinant antigens also have been proved to increase the sensitivity and specificity, compared to native TES. A number of genes encoding TES proteins have been sequenced and cloned which offer the possibility of a more specific diagnostic reagent. Several investigators have reported recombinant antigens (rTES-30 and rTES-120) that are potentially useful for serodiagnosis of toxocariasis in IgG assays. However more validation studies are needed to establish its specificity and sensitivity as a diagnostic reagent.
In the current study, three genes which encode TES-26, TES-32 and TES-120 proteins were selected for cloning and expression. To date, the antigen produced by genes encoding TES-26 and TES-32 have not been reported to have a diagnostic potential for serodiagnosis. However, appearance of a 26 kDa protein band is being used as one of the six serodiagnostic markers for confirmation of human toxocariasis in a commercial TES-based
Western blot IgG kit (Testline, USA); thus we hypothesized that rTES-26 would be a useful diagnostic reagent. TES-32 is a prominent 32 kDa N-glycosylated protein secreted by tissue- stage T. canis parasites and is also being used as one of the six serodiagnostic markers for confirmation of human toxocariasis in the above mentioned Western blot IgG kit. The rTES-
120 has previously been reported to have diagnostic potential for toxocariasis, however, this was produced as insoluble protein using a different expression system (than being used in this study) and the immunoassay developed using the recombinant antigen is based on IgG antibody detection. The rTES-30 recombinant antigen seemed to be highly specific for toxocariasis. However based on discussions with an expert in the field who has used rTES-
30 antigen, the antigen alone was found not to be highly sensitive when tested with serum samples from another part of the world.
The low specificity of the Toxocara serodiagnosis can be improved significantly by employing IgG4 as the secondary antibody since Toxocara-specific IgG4 has been shown to significantly reduce the cross-reaction with antibodies to other helminthic infections
(Wiechinger, 1998; Rahmah et al., 2005, Norhaida et al., 2008). Previous study indicated that IgG4-based immunodiagnosis for toxocariasis produced less false positive than IgG- based immunoassay when tested with serum samples from other parasitic infections
61 (Rahmah et al., 2005; Norhaida et al., 2008). In this study, various antibody classes and IgG subclasses were tested in order to develop the most sensitive and specific assay for diagnosis of toxocariasis. We hypothesized that a panel of recombinant antigens combined with the most appropriate antibody class/subclass will enable the development of a robust, sensitive and specific assay for toxocariasis.
1. 15 Objectives of the study
The goal of this study is to develop recombinant antigen (s)-based test for specific and sensitive serodiagnosis of human toxocariasis. To achieve these goals, specific objectives of the study are as follows:
1. To establish culture of T. canis stage two larvae (L2) for extraction of mRNA for use
in cloning of open-reading frames (ORF) of several Toxocara genes.
2. To clone and express three candidate genes encoding rTES-26, rTES-32 and rTES-
120 recombinant proteins from the mRNA of T. canis.
3. To purify three recombinant antigens of T. canis using affinity purification
techniques.
4. To perform evaluation of the diagnostic value of the recombinant antigens produced
in this study, along with our previously produced rTES30USM.
In this study, T. canis larvae culture was established and used for mRNA extraction. This was followed by cloning of ORF of genes encoding TES-26, TES-32 and TES-120 via reverse-transcriptase-PCR (RT-PCR). The ORF of the genes encoding TES-26 and TES-32 were cloned into GST-tag expression vector while TES-120 was cloned into His-tag expression vector. After the recombinant plasmids have been verified, these plasmids were transformed into BL21 (DE3) expression hosts. Small-scale expressions of these recombinant proteins from selected clones in E. coli were performed to optimize post induction time-points, to study the immunogenicity of recombinant proteins and to check the solubility of the proteins. Subsequently, ELISA assay (s) based on various antibody classes
62 and IgG subclasses using each of the recombinant antigens were tested. The diagnostic utilites of each recombinant antigen and rTES-30USM (previously produced in our laboratory) were further evaluated to determine their sensitivities and specificities using a sera panel comprising Toxocara infections, other related parasitic infections as well as healthy individuals. Finally, an immunoassay using three recombinant antigens in adjacent wells was developed for detection of toxocariasis.
CHAPTER 2
GENERAL MATERIALS AND METHODS
63 2.1 Materials
2.1.1 T. canis worms
Approximately 20 naturally infected puppies and 90 dogs were obtained from different locations in Malaysia from August 2004 to September 2005. This was made possible due to the collaborations and support obtained from the Seberang Prai City Council, Penang; Kota
Bharu City Council, Kelantan; and Veterinary Departments of Kota Bharu, Kelantan and
Kangar, Perlis. These were stray animals that were meant to be 'put down' for public safety.
After being sacrified, they were brought to Veterinary Laboratories at Bukit Tengah
(Penang), Kubang Kerian (Kelantan) and Kangar (Perlis). All the post-mortem procedures employed sterile surgical equipment sets. The small intestines were removed, washed with cold sterile normal saline and subsequently these organs were placed in containers containing sterile normal saline. Each intestine was cut longitudinally and the mucosa was carefully examined for adult worms of T. canis. After collection, the T. canis adult worms
(both male and female) were washed and kept in cold sterile normal saline solution before being taken to the laboratory. The adult male and non-gravid female worms were directly frozen at -80ºC and some were preserved in 10% formalin.
2.1.2 Oligonucleotide primers
Primers used in this study were synthesized by First Base, Malaysia. The primers were PCR grade except for mutagenesis primers which were PAGE-purified grade to ensure maximum quality. These oligonucleotide primers were obtained in readily desalted lyophilized form.
The detailed descriptions of all primers will be discussed further in Chapter 3.
2.1.3 Bacterial strains
64 The E. coli strains used for the cloning and expression studies are listed in Table 2.1. E. coli strains were prepared and maintained following the standard protocols (Sambrook & Russell,
2001) by regularly transferring the colonies onto fresh LB agar plate once a week.
2.1.4 Cloning and expression vectors
The plasmid vector, pCR®2.1TOPO® (Invitrogen, USA) was employed as the cloning vector.
The expression vectors used in this study are pET42a and b (+) (Novagen, Germany) and pPROExTMHTa (Life Technologies, USA). The detailed description of each vector is discussed in Chapter 3.
2.1.5 Human serum samples
A total of 242 serum samples from patients with confirmed parasitic infections, either diagnosed parasitologically, serologically and/or clinically and from healthy individuals were employed in this study. The serum samples were obtained from 30 patients with clinical, haematological and serological evidence of toxocariasis (mostly VLM) which were shown to be seropositive by IgG-ELISA commercial kit (Cypress Toxocara IgG, Belgium),
28 samples from patients with soil-transmitted helminthes (STH) i.e. A. lumbricoides, T. trichiura and/or hookworm, 30 samples from patients with serological diagnosis of amoebiasis, 20 samples from patients with serological diagnosis of toxoplasmosis, 28 samples from patients with B. malayi microfilaremia, 5 samples from patients with strongyloidiasis, 1 sample from patient with Gnathostoma spinigerum in the eye and 100 from healthy individuals with no evidence of helminth infections.
Serum samples were obtained from Scottish Parasite Diagnostic Laboratory (SPDL),
Glasgow, United Kingdom; Hospital Universiti Sains Malaysia (HUSM), Kelantan; Hospital
Universiti Kebangsaan Malaysia (HUKM); University of Malaya, Kuala Lumpur and USM
Table 2.1 E. coli strains employed in this study
65 E. coli strains Genotype Supplier Usage
E. coli TOP10 F-, mcrA, hsdR, lacZ∆M15, Invitrogen, USA non-expression
∆lacX74, recA1, endA1 host
E. coli BL21 (DE3) F-, ompT, hsdS(r-m-), gal, Novagen, Germany expression
dcm (DE3) host
66 Health Centre, Penang, Malaysia. These were previously collected according to the ethical requirements of each institution. The collected sera were aliquoted and stored at -20ºC.
2.1.6 Chemicals, reagents and media
All chemicals, reagents and media employed in this study were electrophoresis, molecular biology or analytical grade and are listed in Appendix A.
2.1.7 Sterilisation
2.1.7.1 Moist heat
The appropriate media were autoclaved at 121ºC, 15 psi for 15 minutes. The media were tested for sterility by incubating at 37ºC overnight to check for contamination, and stored at
4ºC. Distilled and deionized water, micropipette tips, microcentrifuge tubes, glasswares and flasks were also sterilized by this method. These were then placed in 50ºC oven for several hours to dry prior to storage.
2.1.7.2 Membrane filtration
Heat-labile media, solutions and antibiotics were sterilized using disposable syringe filters with pore size of 0.22 µm. All solutions and media for tissue culture were prepared aseptically in a laminar flow safety cabinet class II (Nuaire, USA).
2.1.7.3 DEPC treatment
The glasswares and water used for mRNA isolation were treated with a 0.1% diethyl pyrocarbonate (DEPC) for 24 hours at 37ºC. After removing the DEPC, they were placed in the hot air oven at 180ºC for a minimum of 3 hours to destroy the remaining DEPC.
67 2.1.8 Preparation of common media
2.1.8.1 Luria-Bertani (LB) broth
This media was prepared by dissolving 10 g bacto-tryptone, 5 g yeast extract and 10 g sodium chloride (NaCl) in 800 ml deionized water. The broth was adjusted to pH 7.5 with 3
M sodium hydroxide (NaOH) and was made up to 1 L with deionized water. The broth was then dispensed in 10 ml aliquots into universal bottles and autoclaved. The media was stored at 4ºC until used. Ampicillin or kanamycin (final concentration of 50 and 30 µg/ml, respectively) was added to the broth shortly before used.
2.1.8.2 Terrific broth (TB)
Terrific broth was prepared by dissolving 12 g bacto-tryptone, 24 g yeast extract and 4 ml glycerol in 800 ml deionized water. The volume was made up to 900 ml with deionized water and sterilized by autoclaving. The broth was made up to 1L with salt solution prior to use.
2.1.8.3 Salt solution
Salt solution was prepared by dissolving 125.4 g dipotassium hydrogen phosphate (K2HPO4) and 23.1 g potassium dihydrogen phosphate (KH2PO4) in 800 ml of deionized water. The volume was made up to 1 L with deionized water and sterilized by autoclaving. A 100 ml salt solution was added to 900 ml of Terrific broth shortly before used.
2.1.8.4 LB agar
LB agar was prepared as described above (subsection 2.1.8.1) with addition of 15 g bacteriological agar to the LB broth. The pH was adjusted to 7.5 with 3 M NaOH, made up to 1 L with deionized water and autoclaved. The agar was allowed to cool to 50ºC in a waterbath before pouring into petri dishes (15 to 20 ml). The LB agar was allowed to solidify at room temperature and stored at 4ºC until used.
68 2.1.8.5 LB agar with ampicillin
LB agar was prepared as described above (subsection 2.1.8.4). The agar was allowed to cool to 50ºC in a waterbath before ampicillin (final concentration of 50 µg/ml) was added. The media was poured into petri dishes (15 to 20 ml), allowed to solidify at room temperature and stored at 4ºC until used.
2.1.8.6 LB agar with kanamycin
LB agar was prepared as described above (subsection 2.1.8.4). The agar was allowed to cool to 50ºC in a waterbath before adding kanamycin to a final concentration of 30 µg/ml. The media was poured into petri dishes (15 to 20 ml), allowed to solidify at room temperature and stored at 4ºC until used.
2.1.9 Preparation of common buffers and reagents
2.1.9.1 Phosphate buffered saline (PBS), pH 7.2
This buffer was prepared by dissolving 8 g of NaCl, 0.2 g of potassium chloride (KCl), 1.44 g of disodium hydrogen phosphate (Na2HPO4) and 0.24 g of KH2PO4 in 800 ml deionized water. The pH was adjusted to 7.2 with 1 N hydrochloric acid (HCl) and the volume was made up to 1 L with deionized water. The buffer was then autoclaved and stored at 4ºC.
2.1.9.2 PBS-Tween 20 (PBS-T), 0.05% (v/v)
The washing buffer was prepared by adding 0.5 ml of Tween 20 to 1 L of PBS.
2.1.9.3 Tris buffered saline (TBS)
This buffer was prepared by dissolving 1.58 g of Tris-HCl and 9 g of NaCl in 800 ml deionized water. The pH was adjusted to 7.5 with 1 N HCl and the volume was made up to 1
L with deionized water. The buffer was then autoclaved and stored at 4ºC.
69 2.1.9.4 TBS-Tween 20 (TBS-T), 0.05% (v/v)
This wash buffer was prepared by adding 0.5 ml of Tween 20 to 1 L of TBS.
2.1.9.5 TBS/Tween 20/Triton-X (TBS-TT) (w/v/v)
This buffer was freshly prepared by dissolving 3.15 g of Tris-HCl (20 mM), 29.22 g of NaCl
(500 mM), 0.5 ml of Tween 20 and 2 ml of Triton-X100 in 800 ml deionized water. The pH was adjusted to 7.5 and the volume was made up to 1 L with deionized water.
2.1.9.6 NaOH solution (3M)
This solution was prepared by dissolving 12 g of NaOH pellet in 50 ml of deionized water and the volume was made up to 100 ml with deionized water. The solution was then autoclaved and stored at room temperature.
2.1.9.7 HCl solution (1M)
A 1 M HCl was prepared by mixing 8.28 ml of HCl stock solution (37%) in 91.72 ml deionized water. The solution was then stored in a glass bottle at room temperature.
2.1.9.8 EDTA solution, 0.5 M (pH 8.0)
This solution was prepared by dissolving 18.61 g of EDTA (disodium salt dehydrate) in 80 ml of deionized water by vigorously stirring with magnetic stirrer while the pH of the solution was adjusted to 8.0 with 3 M NaOH. The final volume was made up to 100 ml with deionized water. The solution was then autoclaved and stored at 4ºC.
2.1.9.9 Ampicillin stock solution (100 mg/ml)
One gram of ampicillin was dissolved in 10 ml of deionized water and was sterilized by filtration through 0.22 µm syringe filter. The solution was then aliquoted (1 ml each) into microfuge tubes and stored at -20ºC.
70 2.1.9.10 Kanamycin stock solution (30 mg/ml)
This stock solution was prepared by dissolving 0.3 g of kanamycin in 10 ml of deionized water and was filter sterilized using 0.22 µm syringe filter. The solution was then aliquoted
(1 ml each) into microfuge tubes and stored at -20ºC.
2.1.9.11 Magnesium chloride (MgCl2), 100 mM
This solution was prepared by dissolving 0.02 g MgCl2 in 80 ml deionized water. The volume was made up to 100 ml with deionized water, autoclaved and stored at 4ºC.
2.1.9.12 Calcium chloride (CaCl2), 100 mM
This solution was prepared by dissolving 1.47 g CaCl2 in 80 ml deionized water. The volume was made up to 100 ml with deionized water, autoclaved and stored at 4ºC.
2.1.9.13 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), 20 mg/ml (w/v)
This solution was prepared by dissolving 0.2 g X-gal in 10 ml dimethylformamide, stored as
1 ml aliquots at -20ºC in microfuge tubes (wrapped with aluminium foil).
2.1.9.14 Isopropyl- ß-D- galactopyranoside (IPTG), 800 mM
This stock solution was prepared by dissolving 5 g of IPTG in 26.23 ml of deionized water and was filter sterilized using 0.22 µm syringe filter. The solution was then stored as 1 ml aliquots at -20ºC.
2.1.9.15 Ethanol (70%)
This solution was prepared by mixing 70 ml of absolute ethanol and 30 ml of deionized water. The solution was stored at room temperature.
71 2.1.10 Preparation of reagents for culture of L2 larvae
2.1.10.1 Normal saline [sodium chloride, 0.85% (w/v)]
This solution was prepared by dissolving 8.5 g NaCl in 800 ml deionized water. The volume was made up to 1000 ml with deionized water, autoclaved and stored at 4ºC.
2.1.10.2 RPMI-1640 stock solution
RPMI-1640 medium was prepared according to the manufacturer's instruction (Gibco BRL.,
UK) by dissolving 5.2 g of RPMI-1640 base medium and 1 g of NaHCO3 in 400 ml of deionized water. The pH was adjusted to pH 7.2 and deionized water was then added to make up to 500 ml solution. The medium was sterilized by filtration through 0.22 µm filter and stored at 4ºC. Prior to use, 1 ml of penicillin solution (100 IU/ml), 1 ml of streptomycin solution (100 µg/ml) and 1 ml of fungizone solution (2.5 µg/ml) was added to 100 ml of
RPMI solution.
2.1.10.3 Acid-pepsin solution [Pepsin 1% (w/v), HCl 0.1% (v/v)]
This solution was prepared by dissolving 1.0 g pepsin and 100 µl HCl in 80 ml deionized water. The volume was made up to 100 ml with deionized water, filter sterilized using 0.22
µm syringe filter and stored at 4ºC.
2.1.10.4 DEPC-treated water
One ml of DEPC solution (commercially obtained) was added into 1 L of deionized water, mixed and autoclaved to inactivate the DEPC. This water was used for preparing buffers and solutions for mRNA extraction.
2.1.11 Preparation of reagents for agarose gel electrophoresis
2.1.11.1 Tris-Borate-EDTA (TBE) buffer (5X)
This stock buffer was prepared by dissolving 54 g Tris base, 27.5 Boric acid and 20 ml
EDTA solution (0.5 M) in 800 ml deionized water. The volume was made up to 1000 ml
72 with deionized water and stored at room temperature. One hundred ml of stock solution was diluted with 900 ml of deionized water before used.
2.1.11.2 Ethidium bromide solution (EtBr), 10 mg/ml (w/v)
This stock solution was prepared by dissolving 1g EtBr in 80 ml deionized water. The volume was made up to 100 ml deionized water and stored in a dark bottle at room temperature.
EtBr is a highly carcinogenic substance due to its ability to intercalate in the double helix of DNA. Therefore, all EtBr-contaminated materials were handled with gloves and disposed of properly according to the protocol suggested by Sambrook et al. (1989). The
EtBr waste solution was filtered through Whatman’s paper containing a bed of activated charcoal in order to neutralize the EtBr. The filtrate was then poured down the drain.
2.1.11.3 Agarose gel loading dye
The loading dye was prepared by dissolving 20 g of sucrose and 0.25 g of Orange-G in 30 ml of deionized water. The deionized water was then added to make up to 50 ml. This loading dye was stored as 1 ml aliquots at -20ºC.
2.1.12 Preparation of reagents for SDS-PAGE
2.1.12.1 Resolving gel buffer, pH 9.3
Resolving gel buffer was prepared by mixing 80 ml of solution A (1.5 M Tris/0.4% SDS) and 20 ml of solution B (1.5 M Tris-HCl/0.4% SDS). The buffer was adjusted to pH 9.3 with either solution A or solution B, filtered and stored at 4ºC. Solution A was prepared by dissolving 72.7 g Tris base and 1.6 g SDS in 300 ml deionized water. The final volume was made up to 400 ml with deionized water. Solution B was prepared by dissolving 23.7 g Tris-
HCl and 0.4 g SDS in 80 ml deionized water. The final volume was made up to 100 ml with deionized water.
73 2.1.12.2 Stacking gel buffer, pH 6.8
The stacking buffer was prepared by dissolving 6 g of Tris base and 0.4 g of SDS in 40 ml deionized water. The solution was adjusted to pH 6.8 with 1 N HCl. The final volume was made up to 100 ml with deionized water, filtered and stored at 4ºC.
2.1.12.3 2X Sample buffer
The sample buffer was prepared by dissolving 0.76 g of Tris base, 1.0 g of SDS and 10 ml glycerol in 20 ml deionized water. The solution was adjusted to pH 6.8 with 1 N HCl. The final volume was made up to 50 ml with deionized water, filtered and stored at 4ºC. Prior to use, 2-ß-mercaptoethanol (final concentration of 10%) and bromophenol blue (final concentration of 0.05%) were added to the sample buffer.
2.1.12.4 Ammonium persulfate (AP), 20% (w/v)
This solution was freshly prepared by dissolving 0.04 g ammonium persulfate in 200 µl deionized water. The ammonium persulfate powder was kept in a tightly closed container, stored in a cool and dry place.
2.1.12.5 Running buffer
This buffer was freshly prepared by dissolving 3.0 g Tris base, 14.4 g glycine and 1.0 g SDS in 800 ml deionized water. The pH was adjusted to 8.3. The final volume was then made up to 1 L with deionized water.
2.1.12.6 Coomassie blue stain
This stain was prepared by dissolving 0.8 g Coomassie Brilliant Blue R250 in 100 ml isopropanol. Once dissolved, 260 ml of deionized water was added and stored at room temperature. Ten ml glacial acetic acid (10%) was added to 90 ml of this solution prior to use.
74 2.1.12.7 Coomassie destaining solution
This solution was prepared by adding 100 ml glacial acetic acid and 100 ml methanol to 800 ml deionized water, stored at room temperature.
2.1.13 Preparation of reagents for immunodetection
2.1.13.1 Western blot transfer buffer
This buffer was prepared by dissolving 3.0 g Tris base and 14.4 g glycine in 800 ml deionized water and stored at 4ºC. Two hundred ml methanol (20%) was added to the solution prior to use.
2.1.13.2 Ponceau S stain
This staining solution was prepared by dissolving 0.5 g Ponceau S in 80 ml distilled water.
One ml glacial acetic acid was added and the volume was adjusted to 100 ml with deionized water.
2.1.13.3 Amido black stain
This staining solution was prepared by dissolving 0.1 g Amido Black (Naphtol Blue Black),
10 ml glacial acetic acid and 45 ml 100% methanol in 45 ml deionized water. The volume was adjusted to 100 ml with distilled water and stored at room temperature.
2.1.13.4 Blocking stock solution, 10% (w/v)
This stock solution was prepared according to the manufacturer's instruction by dissolving
27 g of blocking powder (for ELISA) (Roche Diagnostic, Germany) in a final volume of 100 ml deionized water. The stock solution was then aliquoted (10 ml each) into 15 ml tubes and stored at -20ºC. The working solution 1% was prepared by adding 5 ml of stock solution with 45 ml PBS (10X dilution).
75 2.1.13.5 Chemiluminescence substrate (Roche Diagnostics, Germany)
The substrate solutions A and B were brought to room temperature prior to use. The substrate was prepared by mixing prewarmed substrate solution A and starting solution B in a ratio of 100:1. The working solution was incubated for additional 30 minutes at room temperature before use.
2.1.14 Preparation of reagents for histidine-tagged protein purification under
native condition
2.1.14.1 Protease inhibitor cocktail (0.37 mg/ml)
This stock solution was prepared by dissolving 1 tablet of protease inhibitor cocktail in 2 ml of deionized water. This solution was vortexed until completely dissolved and filter sterilized by using a 0.22 µm syringe filter, stored as 1 ml aliquots in microfuge tubes at
-20ºC.
2.1.14.2 Lysozyme stock (10 mg/ml) (w/v)
This stock solution was prepared by dissolving 0.2 g of lysozyme in 20 ml of deionized water. This solution was filter sterilized by using a 0.22 µm syringe filter, aliquoted (1 ml each) into microfuge tubes and stored at -20ºC.
2.1.14.3 DNAse 1 (2500 µg/ml) (w/v)
This stock solution was prepared by dissolving 50 mg/U of DNAse 1 in 10 ml of 20 mM
Tris-Cl and 10 mM MgCl2, pH 8.0. This solution was incubated on ice for 30 mins, mixed well and filter sterilized by using a 0.22 µm syringe filter. The stock solution was then aliquoted (100 µl each) into microfuge tubes and stored at -20ºC.
2.1.14.4 10 mM lysis buffer (containing 300 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (300 mM) and 0.68 g imidazole (10 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
76 M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.5 20 mM wash buffer (containing 300 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (300 mM) and 1.36 g imidazole (20 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.6 30 mM wash buffer (containing 300 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (300 mM) and 2.04 g imidazole (30 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.7 40 mM wash buffer (containing 300 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (300 mM) and 2.72 g imidazole (40 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.8 Elution buffer (containing 300 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (300 mM) and 17.0 g imidazole (250 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with
3 M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
77 2.1.14.9 10 mM lysis buffer (containing 500 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 29.23 g NaCl (500 mM) and 0.68 g imidazole (10 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.10 20 mM wash buffer (containing 500 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 29.23 g NaCl (500 mM) and 1.36 g imidazole (20 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.11 30 mM wash buffer (containing 500 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 29.23 g NaCl (500 mM) and 2.04 g imidazole (30 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.12 40 mM wash buffer (containing 500 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 29.23 g NaCl (500 mM) and 2.72 g imidazole (40 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.13 Elution buffer (containing 500 mM NaCl)
This buffer was prepared by dissolving 5.99 g NaH2PO4 (50 mM), 17.54 g NaCl (500 mM) and 17.0 g imidazole (250 mM) in 800 ml deionized water. The pH was adjusted to 8.0 with
78 3 M NaOH. The volume was adjusted to 1L with deionized water and stored at room temperature.
2.1.14.14 Stripping buffer
This buffer was prepared by dissolving 0.24 g NaH2PO4 (20 mM), 1.75 g NaCl (500 mM) and 3.4 g imidazole (500 mM) in 90 ml deionized water. The pH was adjusted to 8.0 with 3
M NaOH. The volume was adjusted to 100ml with deionized water and stored at room temperature.
2.1.15 Preparation of reagents for GST-tagged protein purification under native
condition
2.1.15.1 GST bind/wash buffer (1X)
The 1X working buffer was prepared according to the manufacturer's instruction by diluting
100 ml of 10X supplied stock (containing 43 mM NaH2PO4, 14.7 mM KH2PO4, 1.37 M NaCl and 27 mM KCl) (pH 7.3) (Novagen, Germany) with 900 ml of deionized water (1:10 dilution).
2.1.15.2 GST elution buffer (10X) (w/v)
The stock 10X GST elution buffer was prepared by dissolving 1 g reduced glutathione in
32.5 ml 10X glutathione reconstitution buffer (containing 500 mM Tris-Cl, pH 8.0)
(Novagen, Germany). The stock solution was stored as 2 ml aliquots at -20ºC to prevent oxidation of the glutathione. The stock solution is stable for 6 months at -20ºC with no more than 5 freeze/thawed cycles. The 1X GST elution buffer was prepared by diluting 1 ml of stock solution with 9 ml of glutathione reconstitution buffer shortly before use.
79 2.1.16 Preparation of reagents for ELISA
2.1.16.1 Coating buffer
This solution was prepared by dissolving 1.465 g of sodium bicarbonate (NaHCO3) and
0.795 g of sodium carbonate (NaCO3) in 450 ml deionized water. The pH was adjusted to 9.6 and the volume final was made up to 500 ml with deionized water, autoclaved and stored at room temperature.
2.1.16.2 Blocking stock solution, 10%
This stock solution was prepared according to the manufacturer's instruction by dissolving
27 g of blocking powder (for Western blot) (Roche Diagnostic, Germany) in 100 ml deionized water. The stock solution was then aliquoted (10 ml each) into 15 ml tubes and stored at -20ºC. The working solution 1% was prepared by adding 5 ml of stock solution with 45 ml PBS (ratio of 1:10).
2.1.16.3 Substrate 2,2’-Azino-bis(3-ethylbenthiazoline-6- sulphonic acid) (ABTS)
This substrate solution was prepared according to the manufacturer's instruction (Roche
Diagnostic, Germany) by diluting 5 ml substrate buffer (stock solution) with 45 ml deionized water (ratio at 1:10). One ABTS tablet (50 mg) was dissolved in this solution to make a final concentration of 1 mg/ml. The substrate was then aliquoted and stored at 4ºC in the 50 ml tube wrapped with aluminium foil to protect it from light. The substrate was brought to room temperature prior to use.
2.2 Methods
2.2.1 Isolation and cultivation of T. canis eggs
The in vitro cultivation of the second stage larvae was performed according to the method of
Maizels et al. (1984) and Chung et al. (2004) with some modifications.
Approximately 100 gravid female worms were dissected for unembryonated eggs.
Each gravid adult female worm was dissected using sterile blade and the lower one-third of
80 the uterus (where the proportion of unembryonated eggs is usually higher than in the remainder of the uterus) was isolated. The fertile eggs were removed and the eggs material
(eggs suspension containing other worm parts) was digested in acid-pepsin solution for 30 minutes. The mixture was then passed through a fine sieve to remove the unwanted materials. The eggs suspension was washed twice with sterile cold normal saline by centrifugation at 5 000 x g for 10 minutes. Subsequently, the eggs were incubated in 50 ml
2% neutral formalin in a sterile 250-ml flask for 14 days at 30ºC with occasional inspection.
A little sample was taken every three days for examination of the development of embryonated eggs under the microscope (Nikon Eclipse 80i, Japan).
Subsequently, the fully embryonated eggs were washed for five times in sterile cold distilled water by centrifugation at 500 x g for 10 minutes wash to get rid of the formalin.
Then a 10% eggs suspension was made in sterile cold distilled water. An equal volume of ~
4% sodium hypochlorite solution was added to the 10% eggs suspension. The mixture was mixed on a shaker at room temperature for 30 minutes in order to decoat the embryonated eggs. A hand tissue homogenizer (B.Braun, USA) was then used to discrupt the decoated eggs. A little sample was taken and was examined under a microscope to check whether the eggs shell was broken. Subsequently, the eggs suspension was washed 10 times as above until all traces of chlorine had been removed.
The pellet containing larvae was resuspended in 100 ml RPMI-1640 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and fungizone (2.5 µg/ml) and transferred into four sterile petri dishes. A mixture of 5% CO2 and 95% N2 was bubbled through the suspension at 37ºC in the carbon dioxide (CO2) incubator (Thermo Electron
Corp., USA) under sterile conditions. After one hour, the mixture was transferred to a modified Baermann’s apparatus made up of two layers of sterile gauze on a steel sieve
(Figure 2.1). The motile larvae that had migrated through the gauze were collected from the bottom of the Baermann’s apparatus after 8 hours. The larvae were centrifuged at 5 000 x g for 30 minutes. The supernatant was removed and the pellet containing T. canis L2 larvae was washed twice with sterile cold distilled water by centrifugation as described above.
81 2.2.1.1 Storage of T. canis larvae
RNA stabilization is a prerequisite for reliable gene expression analysis. Immediate stabilization of RNA in biological samples is necessary because, directly after sample harvesting, changes in the gene expression pattern occur due to specific and nonspecific
RNA degradation. RNAlater RNA Stabilization Reagent (Qiagen, USA) penetrates the sample by diffusion into individual cells and into the surface layer and outer portions of solid tissues and protects the cellular RNA.
The preparation of the larvae sample prior to storage was performed as described by the manufacturer (Qiagen, USA). The pellet containing T. canis L2 larvae to be stabilized
(from 2.2.1) was quickly weighed. Ten volumes of RNAlater RNA Stabilization Reagent (or approximately 10 µl/1 mg of larvae) was immediately added to the larvae samples and was kept at 4ºC overnight. The next day, the tube containing the solution was transferred to
-80ºC.
2.2.2 Plasmid extraction
Plasmid was extracted using Wizard®Plus SV Minipreps DNA Purification System
(Promega, USA) following the manufacturer's instructions. Briefly, bacterial cultures were lysed and the lysates was cleared by centrifugation. The cleared lysates was then applied to the Wizard® SV Minicolumn where the plasmid DNA adsorbs to the silica-gel membrane.
Impurities were washed away and pure DNA was eluted in distilled water.
A single, well-isolated bacterial colony containing the desired plasmid was inoculated in 10 ml LB broth (containing the desired antibiotic) and incubated overnight at
37ºC in an incubater shaker (Thermo Electron Corp., USA). The cultures were then harvested by centrifugation at 10 000 x g for 5 minutes. The pellet was thoroughly resuspended in 250 µl of Cell Resuspension Solution by pipetting up and down.
Immediately, 250 µl of Cell Lysis Solution was added and mixed by gently inverting the tube 4 times. The solution was incubated for 1 to 5 minutes until the cell suspension became
82 Retort stand
Two layers of gauze
Glass funnel
Clamp
Figure 2.1 Modified Baermann’s apparatus for collection of T. canis larvae
83 clear. Subsequently, 10 µl of alkaline protease was added, mixed by gently inverting the tube
4 times and incubated for 5 minutes at room temperature. The alkaline protease activates endonucleases and other proteins released during the lysis of the bacterial cells. The solution was neutralized with 350 µl of neutralization solution and the tube was immediately inverted
4 times. Cellular debris was pelleted by centrifugation at 14 000 x g for 10 minutes.
After centrifugation, the supernatant was carefully transferred to the spin column placed into a 2 ml collection tube without disturbing the white precipitate. The tube was centrifuged at 14 000 x g for 1 minute. The flow-through collected in the collection tube was discarded. The plasmid DNA bound to the membrane was washed with 750 µl of Wash
Solution and the tube was centrifuged as previously described. The flow-through was discarded, 250 µl of Wash Solution was added and the tube was further centrifuged at 14 000 x g for 2 minutes to remove residual Wash Solution. Finally, the spin column was transferred to a sterile 1.5 ml tube and the plasmid DNA was eluted by adding 100 µl of sterile distilled water. The tube was allowed to stand for 1 minute and then centrifuged at 14 000 x g for 1 minute. The concentration and purity of the purified plasmid DNA was measured using spectrophotometric absorption at 260 and 280 nm as described below (section 2.2.3). The extracted plasmid DNA was stored at -20ºC for further use.
2.2.3 Quantification of nucleic acids
The concentration and purity of nucleic acids in aqueous solution was determined by spectrophotometric measurement at wavelengths of 260 and 280 nm using Biophotometer
(Eppendorf, Germany). The Biophotometer was blanked with 50 µl of sterile deionized water in a cuvette. One µl of the nucleic acid sample was mixed well with 49 µl of deionized water in a cuvette. The cuvette was then placed into a Biophotometer machine and read at
260 and 280 nm. Pure nucleic acid has an A260/280 nm ratio of >1.8 (for DNA) and 1.8-2.1
(for RNA) (Sambrook & Russell, 2001). The concentration of nucleic acids was calculated using the formula of A260 of 1 is equivalent to 50 µg/ml for double stranded DNA and 40 µg/ ml for single stranded RNA.
84 2.2.4 Polymerase chain reaction (PCR)
The screening of positive recombinant clones was performed according to the method described by Sambrook & Russel (2001). The preparation of PCR master mix was carried out in a laminar flow cabinet. All PCR reagents were allowed to thaw on ice before brief centrifugation. The PCR master mix was prepared in a final volume of 20 µl which consisted of water (DNAse & RNAse-free, Sigma, USA), 1X PCR buffer, 2.5 mM MgCl2, 200 µM of dNTPs mix, 20 pmol/µl of each forward and reverse primers, DNA template and 1U/µl of
Taq DNA polymerase. The master mix was transferred into sterile thin-walled flat-capped
PCR tubes and briefly centrifuged. A negative PCR control was included in each run, which consisted of all the above mixtures except the DNA template.
The PCR tubes were then placed in a thermal cycler (MJ Research, USA). The amplification was performed for 30 cycles at 94ºC for 1 minute, 55ºC for 1 minute and 72ºC for 1 minute, followed by a final extension at 72ºC for 10 minutes. The PCR products were then analyzed by agarose gel electrophoresis as described below to confirm the presence of specific insert (section 2.2.5) and visualization under UV transilluminator.
2.2.5 DNA agarose gel electrophoresis
Agarose gel electrophoresis was used to verify the presence of PCR products, plasmid DNA and inserts. The concentration of agarose gel used in this study was 1.0%. To prepare a 1.0% agarose gel, 0.5 g of agarose was heated in 50 ml of 0.5x TBE in microwave oven for 2 minutes until completely dissolved and allowed to cool down to approximately 50ºC to 60ºC under running water with continuous mixing. Subsequently, 1 µl of ethidium bromide solution (10 mg/ml) was added and mixed gently before pouring the mixture into the casting tray (Bio-Rad, USA). A suitable comb(s) was placed in the casting tray and the gel was allowed to harden for 30 to 60 minutes. After the agarose gel was solidified, the comb was carefully removed. The gel was completely submerged into the electrophoresis tank containing 0.5 x TBE. The samples were then mixed with 6 x loading buffer at the ratio of
1:5 (1 µl of loading buffer for every 5 µl of sample) and loaded into the wells on the agarose
85 gel. Five µl of the molecular weight markers such as 100 bp DNA ladder and/or 1 kb DNA ladder (Fermentas, USA) were loaded into the adjacent wells to determine the size of the
DNA. Electrophoresis was then run at 80 volts for approximately 60 to 80 minutes. The
DNA bands were visualized under UV light transilluminator and photographed using Gel
Documentation system (Syngene, USA).
2.2.6 Purification of PCR products
The DNA fragment was recovered from agarose gel with "Perfectprep Gel Cleanup" kit
(Eppendorf, USA) using the protocol described by the manufacturer. PCR product purification allows the removal of excess primers, unincorporated dNTPs and salts from the
PCR reaction mixture, proteins, agarose and organics.
The DNA fragment resolved on a TBE agarose gel was excised under UV light transilluminator (Biorad, USA), weighed and 3 volumes of binding buffer was added for every 1 volume of the slice (1 mg of weight equals 1 µl of volume). The mixture was then incubated in a 50ºC waterbath for 10 minutes, and was vortexed every 2 to 3 minutes to dissolve the gel. After the gel slice was completely dissolved, a volume of isopropanol equal to the original gel slice volume was added and was mixed by pipetting up and down. The sample (up to 800 µl) was then added to the spin column attached to 2-ml collection tube.
The tube was centrifuged at 8,000 x g for 1 minute. The filtrate was discarded and the bound
DNA was washed with 750 µl of diluted wash buffer and centrifuged as above. The filtrate was discarded; the spin column was attached to a new collection tube and was centrifuged as above to remove any residual diluted wash buffer. The spin column was then placed in a new
2-ml collection tube and 30 µl of water (DNAse and RNAse-free, Sigma, USA) was added to the center of the spin column. Finally, the column was centrifuged as above to elute out the purified DNA. The concentration of the purified DNA was quantified using a biophotometer as described in section 2.2.3.
86 2.2.7 Preparation of competent cells
Competent cells are cells that have been chemically treated to allow its cell wall to be permeated by plasmids. Competent cells were prepared by CaCl2 method as described by
Sambrook & Russell (2001). A single colony of E. coli TOP 10 strain was inoculated in 5 ml of LB broth and incubated overnight at 37ºC, 200 rpm. The overnight culture (100 µl) was inoculated in 50 ml LB broth in a 250 ml flask, incubated in incubator shaker at 37ºC, 200
7 8 rpm until OD600 reached approximately 0.4 (mid log phase with 10 to 10 cells/ml). The culture was then chilled on ice for 10 minutes with occasional swirling to stop the growth.
Bacterial cells were collected by centrifugation at 3,000 x g for 10 minutes at 4ºC.
Subsequently, the pellet was resuspended in 6 ml pre-cooled sterile 100 mM MgCl2 with gentle mixing. The mixture was incubated on ice for 1 hour, and then centrifuged as above.
The pellet was resuspended in 300 µl of pre-cooled 100 mM CaCl2 and the sterile glycerol was added to a final concentration of 15%. The competent cells were aliquoted (100 µl per tube) into pre-cooled sterile 1.5 ml tubes on ice and quickly stored at -80ºC until needed.
2.2.8 Transformation of plasmid into E. coli TOP10 competent cells
Transformation of plasmid into the competent cells was performed as described by
Sambrook & Russell (2001). The competent cells were removed from -80ºC and thawed on ice for 5 minutes. The cells were mixed by gently tapping the tubes 1-2 times.
In each experiment, a positive control was included to measure the efficiency of transformation and a negative control was included to check for the possibility of contamination. For the positive control, one µl of the test plasmid was used, whereas, one µl of sterile distilled water was used instead of plasmid for the negative control.
One µl of the purified plasmid DNA/5 µl of TOPO cloning reaction/ligation reaction was added to the tubes containing cells and gently stirred with pipette tip. The tubes were incubated on ice for 30 minutes and heat-shocked for exactly 40 seconds in a 42ºC water bath without shaking. The tubes were placed back on ice immediately for 2 minutes.
Following that, 250 µl of room temperature LB broth was added. The tubes were then
87 shaken at 37ºC, 200 rpm for 1 hour in an incubator shaker to allow the bacteria to recover and express the antibiotic marker encoded by the plasmid. For blue/white screening, 40 µl of 40 mg/ml X-gal was spread on each LB/ampicillin plate and incubated at 37ºC until ready to use. After 1 hour, 50 µl of the transformed cells from the test, positive and negative controls were plated onto the pre-warmed LB/ampicillin plates. The agar plates were incubated overnight at 37ºC. The next day the white colonies were screened with PCR method (described in section 2.2.4). The competent cells were not contaminated if there was no growth on the LB/ampicillin agar plated with the negative control preparation.
2.2.9 Transformation of plasmid into E. coli BL21 (DE3) competent cells
One µl of the purified plasmid DNA was added to the tubes containing cells and stirred gently with pipette tip. The tubes were incubated on ice for 5 minutes and heat-shocked for exactly 30 seconds in a 42ºC water bath without shaking. The tubes were placed back on ice immediately for 2 minutes. Next, 250 µl of room temperature SOC medium (commercially available) was added. The tubes were then incubated at 37ºC, 250 rpm for 30 minutes
(kanamycin-resistant plasmid) and 1 hour (ampicillin-resistant plasmid). After incubation, 50
µl of the cell suspension from the test, positive and negative controls were plated onto the
LB/kanamycin or LB/ampicillin plates. The agar plates were incubated overnight at 37ºC.
2.2.10 Long-term storage of recombinant plasmid
Glycerol stocks of the pET and pPROExHT recombinant plasmids were prepared for long- term storage. High glycerol concentration (>10%) may lead to plasmid instability (Sambrook
& Russell, 2001).
A single colony was inoculated in 10 ml LB broth containing appropriate antibiotics
(30 µg/ml kanamycin for pET recombinant and 100 µg/ml ampicillin for pPROExHT recombinant). The broth was incubated at 37ºC at 200 rpm until the OD600 reached approximately 0.6. Subsequently, 900 µl of the cells were added to the tube, followed by addition of 100 µl of 80% glycerol (final concentration of 9%). The solution was mixed well
88 by inverting the tube several times and stored at -80ºC. The transformed cells were recovered by melting a few microliters from the surface and streaking on LB agar plates with appropriate antibiotics.
2.2.11 Protein analysis
2.2.11.1 Determination of protein concentration
The protein concentration was determined with Bradford dye-binding method (Bradford,
1976) by using Bio-Rad protein assay kit (BioRad, USA). Briefly, 1 ml of Bio-Rad protein assay stock solution was mixed with 4 ml of PBS, pH 7.2 to make the working solution. In a microtiter plate, 200 µl of the working solution was added to 10 µl of samples (in duplicate) or protein standard [bovine serum albumin (BSA) standards of 100 to 1000 µg/ml]. The BSA standards were prepared in serial dilution as shown in Table 2.2. The solution was mixed well and absorbance was measured at 595 nm using ELISA reader (TECAN Sunrise,
Austria). The relative measurement of protein concentration was determined by comparing the protein absorbance to a standard curve graph.
2.2.12 Protein analysis by SDS-PAGE
SDS-PAGE is an analytical electrophoretic method used to analyse proteins based on the differences in molecular size. SDS is an anionic detergent that denatures protein and creates a negatively charged protein. Proteins are separated based on molecular size, in which small molecules go through the polyacrylamide gal faster than larger ones in an electric field.
SDS-PAGE was performed according to the discontinuous method (Laemmli, 1970) using
BioRad Mini-Protean II electrophoresis cell (Bio-Rad, USA).
The resolving (separating) gel (10%) was prepared as follows: 2.5 ml of 30% acrylamide/0.8% bisacrylamide, 1.875 ml resolving gel buffer, and 3.125 ml distilled water,
50 µl of 20% ammonium persulfate and 10 µl TEMED. The mixture was mixed well and immediately poured into the assembled minigel electrophoresis cell (Bio-Rad, USA).
Immediately, 1 ml of distilled water was slowly added onto the top of the resolving gel to
89 Table 2.2 Preparation of BSA standards
Concentration of Stock BSA Deionized water (µl) BSA standards (µg/ml) (1000 µg/ml) (µl)
100 100 900
200 200 800
300 300 700
400 400 600
500 500 500
600 600 400
700 700 300
800 800 200
900 900 100
1000 1000 -
90 make an even surface and allowed to polymerize for 45 minutes. Once polymerized, the
4.5% stacking gel was prepared as follows: 0.325 ml of 30% acrylamide/0.8% bisacrylamide, 0.625 ml stacking gel buffer, and 1.525 ml distilled water, 25 µl of 20% ammonium persulfate and 10 µl TEMED. The solution was mixed well and immediately poured on top of the resolving gel. The sample well comb was immediately inserted between the glass plates without trapping air bubbles under the well. The stacking gel was allowed to polymerize for another 45 minutes. Upon polymerization, the sample comb was carefully removed from the stacking gel. The gel was placed into the running chamber of the electrophoresis apparatus containing running buffer.
The protein samples were mixed with 2X SDS-PAGE sample buffers at a ratio of
1:1 and boiled for 10 minutes. Subsequently, the samples were centrifuged at 12 000 x g for
5 minutes. A total protein concentration of 100 µg of each sample was loaded per lane. The protein molecular weight marker (Fermentas, USA or Amersham BioSciences, Sweden) was included in the first lane. Electrophoresis was carried out at constant voltage of 100 volts until the bromophenol blue marker reached 1 cm from the bottom of the gel (approximately
80 minutes). Following electrophoresis, the gel was soaked in the Coomassie Blue staining solution for one hour, followed by overnight destaining in the destaining solution. The gel was then photographed using Bio-Imaging System (Syngene, USA) and dried using GelAir
Dryer (Bio-Rad, USA) for long-term storage.
2.2.13 Determination of immunogenicity of the expressed protein
2.2.13.1 Electrophoretic transfer of proteins to membrane (Western
blotting)
The SDS-PAGE gel was run according to the procedure as previously described in section
2.2.12. The protein was then electroblotted from the gel to nitrocellulose membrane
(Osmonic, USA) by semidry transfer using a Trans-blot®SD Semidry Transfer Cell (Bio-
Rad, USA).
91 After running the gel, filter papers and pre-cut nitrocellulose membrane were incubated in cold transfer buffer for at least 15 minutes. The pre-soaked nitrocellulose membrane was placed on a piece of pre-soaked filter paper, followed by gel which was placed on the nitrocellulose membrane. Next, a second piece of pre-soaked filter paper was placed over the membrane. The air bubbles were removed by rolling over the top using glass rod. The transfer process was performed at 25 mA for 30 minutes. The nitrocellulose membrane was then stained with Ponceau S for 1 minute to check the success of the protein transfer, marked and washed with distilled water several times.
2.2.13.2 Immunoreactivity study of the fusion proteins by Western blot analysis
After the protein has been transferred to the membrane, the membrane was washed with TBS
(3 times for 10 minutes each) and blocked with 1% blocking solution for 1 hour at room temperature on a shaker. Subsequently, the membrane was washed with TBS for 3 x 10 minutes.
2.2.13.2.1 Detection of histidine-tagged protein
The membrane was then cut into strips, followed by incubation with mouse monoclonal anti- histidine-HRP, His-3 probe (SantaCruz, USA) (1:300 dilutions in TBS) at room temperature for 3 hours. The strips were thoroughly washed with TBST for 6 times, 10 minutes each and ready to be developed (refer to subsection 2.2.13.2.4).
2.2.13.2.2 Detection of GST-tagged protein
The membrane then was cut into strips, followed by incubation with GST, monoclonal mouse antibody (IgG1) (Novagen, Germany) (1:10,000 dilution in TBS) at room temperature for 1 hour. The strips were washed with TBSTT for 3 times, 5 minutes each. After washing, the strips were incubated with secondary antibody (monoclonal anti-mouse IgG-HRP)
(Novagen, Germany) (1:5000 dilution in TBS) for 1 hour at room temperature on the shaker.
92 The strips were thoroughly washed as above to avoid non-specific background and ready to be developed (refer to subsection 2.2.13.2.4).
2.2.13.2.3 Detection of specific IgG4 antibody
The membrane then was cut into strips, followed by incubation with serum samples as primary antibody (1:100 dilution in TBS) at 4ºC, overnight. The next morning, the strips were washed with TBST for 4 times, 10 minutes each. After washing, the strips were incubated with secondary antibody (monoclonal mouse anti-human IgG4-HRP) (1:2000 in
TBS, Zymed, USA) for 30 minutes at room temperature on the shaker. The strips were thoroughly washed as above to avoid non-specific background and ready to be developed
(refer to subsection 2.2.13.2.4).
2.2.13.2.4 Development of Western blot using chemiluminescence substrate
The strips were developed in the dark room using BM Chemiluminescence Substrate (Roche
Diagnostics, USA). First, the strips were placed in X-ray cassette (Sigma, USA) and overlaid with BM chemiluminescence blotting substrate-POD. A saran wrap was overlaid on top of the wet strips, followed by an X-ray film (Kodak, USA) on top of it. The exposure time was varied, usually between 30 sec to 10 minutes, depending on the intensity of the signal. The film was then immersed into a solution of developer (Sigma, USA) for a few seconds, rinsed with tap water and fixed with a fixer (Sigma, USA) for at least 1 minute. After rinsing with water, the developed film was air-dried.
2.2.14 Expression of the recombinant proteins
For expression study, the recombinant plasmids were transformed into competent E. coli
BL21 (DE3) expression host as described in subsection 2.2.9. A single colony from freshly subcultured plate was inoculated in 10 ml of TB with appropriate antibiotics (ampicillin resistant for recombinant pPROExHT and kanamycin resistant for recombinant pET). The culture was incubated overnight at 30ºC, 180 rpm. The next morning, 100 µl of the overnight
93 culture was inoculated in a 500 ml flask containing 100 ml TB broth with ampicillin or kanamycin. The cells were grown at 37ºC, 200 rpm until the OD600 reached approximately
0.5. Expression of the target genes were induced with 1 mM IPTG at lower temperature of
30ºC, 200 rpm for 3-5 hours. After incubation, the cells were harvested by centrifugation at
10 000 x g, 4ºC for 15 minutes. The cell pellet was then stored at -80ºC for further use.
Optimization of the induction conditions for each recombinant protein will be discussed in
Chapter 3.
2.2.15 Fast Protein Liquid Chromatography (FPLC) protein purification
FPLC protein purification was performed using AKTATMprime (Amersham BioSciences,
Sweden). The matrix used in this study was HisTrap column containing precharged Ni
SepharoseTM High Performance. Ni SepharoseTM High Performance consists of highly cross- linked agarose beads to which a chelating group has been immobilized. The metal ion nickel
(Ni2+) has been then coupled to the chelating matrix. The binding capacity of the resin is protein-dependent and lies between 5 -10 mg of histidine-tagged protein for every ml medium. The 1/16" 50 ml superloop (Amersham BioSciences, Sweden) was used for sample volume up to 50 ml. It was assembled according to the protocol described by the manufacturer.
2.2.15.1 Preparation of the AKTATMprime
The AKTATMprime machine was prepared and washed according to the protocol described by manufacturers as follows. First, the machine was washed by performing "System Wash
Method" with 20% ethanol, deionized water and wash buffer containing 20 mM imidazole.
The bottom cap of the HisTrap HP 1 ml column (GE Healthcare, Sweden) outlet was removed. After performing "System Wash Method", the column was attached to the machine. Subsequently, the column was washed by performing "Manual Run" with 3-5 column volume (CV) of deionized water and finally equilibrated with at least 5 CV of wash buffer containing 20 mM imidazole, with a flow rate of 1.0 ml/min.
94 At the end of run, the column was disconnected from the AKTATMprime machine, the bottom cap was re-attached and stored at 4ºC. At the end of the process, the AKTAprime machine was washed by performing "System Wash Method" with deionized water and 20% ethanol.
2.2.15.2 Stripping and recharging of the resin
The resin need to be stripped and recharged after 5 - 7 rounds of purifications of the same protein. The resin in the column was stripped and recharged as described below to remove nickel ions and other contaminating protein bound to the resin.
1. Five to ten column volumes of stripping buffer to remove nickel ions.
2. Five to ten column volumes of binding buffer.
3. Five to ten column volumes of deionized water.
4. 0.5 ml of 0.1 M nickel sulphate.
5. Five column volumes of deionized water.
6. Five column volumes of binding buffer (to adjust pH).
7. Five column volumes of 20% ethanol for storage of the resin.
95 CHAPTER 3
CLONING AND EXPRESSION OF ORF OF THE GENES ENCODING
TES-26, TES-32 AND TES-120 IN E. coli
3.1 Introduction
The goal of the study described in this chapter is to construct the recombinant plasmids in order to generate recombinant rTES-26, rTES-32 and rTES-120 proteins. The DNA cloning experiments were performed via RT-PCR and the ORF of the genes encoding TES-26 and
TES-32 were expressed into pET42 expression system (Novagen, Germany) whilst the ORF of the gene which encodes for TES-120 antigen was expressed into pPROExHT expression system (Life Technologies, USA).
A number of genes encoding TES proteins have been previously sequenced, cloned and expressed (Maizels et al., 2000; Maizels et al., 2006). TES-30 corresponding to 30 kDa from TES secreted by infective larvae has been succesfully cloned and expressed in bacterium E. coli (Yamasaki et al., 1998; Norhaida et al., 2008). Tc-muc-1 which encodes the larval TES-120 recombinant antigen has also been previously cloned and expressed in both bacteria E. coli and methylotropic yeast Pichia pastoris but using different expression vectors (Fong et al., 2003; 2004). The gene encoding rTES-26 protein was previously cloned and expressed in pET15b (histidine-tagged vector) and pMAL (maltose-binding protein).
In this study, pET42 system was used for expression of the rTES-26 and rTES-32 recombinant proteins in E. coli, and thus is different from the previously reported expression of rTES-26 recombinant protein using pET15 and pMAL. The pET-42 series is designed for cloning and high-level expression of peptide sequences fused with the 220 amino acid
GST•TagTM protein. The pET-42a-c (+) vectors incorporates the schistosomal GST coding sequence as a fusion partner. The GST•Tag sequence has been reported to enhance the
96 production of recombinant proteins and in some cases the solubility of the fusion proteins
(Smith, 1998). When expressed in the soluble form, GST•Tag fusion proteins can be purified with immobilized glutathione.
The pPROExHT prokaryotic expression system is designed for the expression of foreign proteins in E. coli whereby the gene of interest is cloned into the multiple cloning sites (MCS) of either pPROExHTa, pPROExHTb or pPROExHTc. In a recent study by our group, this vector has been successfully used to express the rTES-30USM recombinant antigen in our laboratory (Norhaida et al., 2008). In this study, pPROExHT expression system was used for expression of the TES-120 recombinant protein, thus different from the previously reported expression of this recombinant protein using pTrcHis2 and yeast (Pichia pastoris) expression systems. Upon expression, the 6 histidine sequence (His)6 tag is located at the amino terminus of the fusion protein. The histidine sequence has a strong affinity for
Ni-Sepharose resin matrix, making it simple to purify the desired protein.
The flowchart of the experimental design in this chapter is shown in figure 3.1. Due to the nature of the experiments in this chapter, it was deemed best to combine methodology and results in order to allow for better flow of the presentation of the write-up.
97 Isolation of T. canis poly(A)+ RNA
Oligonucleotides design
Construction of the recombinant plasmids
RT-PCR of ORF of the genes encoding TES-26, TES-32 and TES-120 using ‘gene’-specific primers
Cloning of RT-PCR product into PCR2.1 TOPO vector
Verification of positive clones (1) PCR screening using both ‘gene’-specific primers; in tandem with vector-specific primer (external) and ‘gene’-specific primer (internal)
(2) Restriction endonuclease digestion
(3) DNA sequencing and sequencing analysis
TES-26 TES-32 TES-120
PCR-based site-directed mutagenesis
DNA sequencing and sequencing analysis
Subcloning into Subcloning into pET42 expression vector pPROExHT expression vector
Restriction enzyme digestion
Continuation on page 75
98 Figure 3.1 Strategy for cloning and expression of ORF of T. canis genes
Ligation of digested insert into expression vector
Transformation into non-expression host (TOP10)
• PCR screening using vector-specific primer (external) and ‘gene’-specific primer (internal) • DNA sequencing and sequencing analysis
Transformation into expression host [BL21 (DE3)]
Recombinant protein expressions
Small-scale expression • Optimization of post-induction time-point
SDS-PAGE analysis
Immunoreactivity study by Western blot analysis for the detection of vector-encoded fusion partners (His-tag and GST-tag)
Figure 3.1 (cont.) Strategy for cloning and expression of ORF of T. canis genes
99 3.2 Methodology and results
3.2.1 Isolation of T. canis poly(A)+ RNA
The mRNA was extracted from T. canis L2 larvae (previously cultured as described in section 2.2.1) using Straight A’s mRNA Isolation System in accordance with the manufacturer’s procedure (Novagen, Germany).
The larvae (kept in RNAlater Stabilization Solution) were removed from the -80ºC and quickly recovered by using membrane filtration technique with pore size of 20 µm. The membrane that contains larvae was immediately placed into 50 ml tube containing 12.5 ml of lysis buffer, gently vortexed to recover L2 larvae and the membrane was quickly removed using a sterile, RNAse-free forcep which was cleaned with RNAseAWAY solution (Qiagen,
USA). Dithiothreitol (DTT) was quickly added to the larvae tissue to a final concentration of
10 mM. The tissue sample was then homogenized using a hand homogenizer (B.Braun,
Germany) until no large tissue fragments were visible (under the microscope 400x magnification) and the mixture appeared homogeneous in colour and consistency. The insoluble material was then removed by centrifugation at 9,000 x g for 15 minutes at room temperature.
During centrifugation, the Magnetight particles (Novagen, Germany) were prepared as follows: One ml of Magnetight Oligo (dT) Particles (10 mg/ml stock) was pipetted into a sterile 1.5 ml tube. Using the Separation Stand (Novagen, Germany), the particles were captured and the supernatant was decanted. One ml of wash buffer was added per 10 mg particles (1 ml/10 mg) and gently vortexed until the particles were fully resuspended. The particles were again captured with the Separation Stand and supernatant was decanted. The washed particles were fully resuspended in one ml wash buffer.
After centrifugation, the tissue lysate supernatant was gently decanted into a sterile conical tube; the washed particles (above) were added and gently mixed by inversion until particles were fully distributed. The mixture was incubated at room temperature for 5 minutes. The particles from the solution were captured using the Separation stand and the
100 supernatant was decanted. The particles were washed with 6 volumes of wash buffer and particles captured. The washing step was repeated twice. One ml of wash buffer was added and the particles were transferred to a new tube. With the tube in the Separation Stand, the supernatant was removed as much as possible, including any residual liquid in the cap.
Subsequently, 0.5-ml of nuclease-free water was added to the particles, resuspended by briefly vortexing and incubated at 60ºC in a water bath for 10 minutes to elute mRNA. The particles were then captured and the supernatant was transferred to a new tube.
Following that, 2 µl of glycogen (10 mg/ml stock) and 50 µl of sodium acetate (3M) were added to 0.5-ml elution from above step, followed by addition of 331 µl isopropanol.
The mixture was vortexed and immediately centrifuged at 14,000 x g for 10 minutes. The supernatant was decanted and the pellet which contains mRNA was washed with 500 µl of
70% ethanol, and centrifuged at 14,000 x g for 5 minutes. The ethanol was completely removed and air-dried for 15 minutes. Finally, the mRNA pellet was completely resuspended in 25 µl nuclease-free water. The yield/concentration and purity of mRNA sample was then quantified by spectrophotometric measurement at wavelengths of 260 and 280 nm using
Biophotometer (Eppendorf, Germany) as described in subsection 2.2.3. The mRNA sample was stored as 5 µl aliquots at -80ºC to prevent degradation until further use for RT-PCR.
The concentration of mRNA at the first preparation was too diluted and could not be measured by biophotometer, however, in the second preparation the concentration was 1.6
µg/µl. The purity of the mRNA was 2.0 (ratio of A260: A280 values should fall in the range of
1.8 – 2.1) for both of the preparations. Due to the low concentration of mRNA, the denaturing agarose gel electrophoresis could not be performed to check the integrity of mRNA sample.
3.2.2 Oligonucleotides design
A pair of primer for each ORF of the gene to be used in RT-PCR was designed based on the nucleic acid sequence (complete cds) of each T. canis excretory-secretory antigen obtained from the National Center For Biotechnology Information (NCBI), National Institute of
101 Health (NIH) Genbank webpage (www.ncbi.nlm.nih.gov) (see Appendixes B), following the guidelines by manufacturer (Stratagene, USA) (Table 3.1). In designing the best primers for higher efficiency amplifications for this study, primers were analyzed and optimized with application softwares, OligoPerfectTM Primer Designer and Vector NTi (Invitrogen, USA).
Primers used in this study were synthesized by First Base, Malaysia. The primers were PCR grade except for mutagenesis primers which were PAGE-purified grade to ensure maximum quality and increase mutation efficiency. These oligonucleotide primers were obtained in readily desalted lyophilized form. Upon arrival, the primers were briefly centrifuged and dissolved in water (DNAse & RNAse-free, Sigma, USA) to a final concentration of 200 pmol/μl. Then, the primers were incubated at 4ºC overnight on a roller
(MacsMix, Miltenyi Biotech, Germany). These stock primers were then aliquoted and stored at -20ºC. Working solution of the primers for PCR was prepared by diluting the stock primers (in water) to working concentration of 20 pmol/μl.
The sense (forward) primers were designed to encode a start codon of the gene. The antisense (reverse) primers were designed to encode a stop codon. Based on the location of the sense and antisense primers, a PCR product size anticipated for each gene is shown in
Table 3.2.
3.2.2.1 Mutagenic oligonucleotide primers
The mutagenic oligonucleotide primers were designed individually according to the desired mutation. Both forward and reverse mutagenic primers contained the desired mutation and annealed to the same sequence on opposite strands of the plasmid. The desired mutation should be in the middle of the primer with ~ 10-15 bases of correct sequence on both sides.
102 Table 3.1 The details of the primers used in this study
Primers Purpose Sequence (5’ to 3’) Length (mer) Reference TES-26F RT-PCR of ORF of gene 5’ – CAC CAT GTC AGT TGT ACA CAA AGC TTG C – 28-mer - encoding TES-26 3’ TES-26R RT-PCR of ORF of gene 5’ – TTA GGC CTG CGA TCG ATA GA -3’ 20-mer - encoding TES-26 TES-32F RT-PCR of ORF of gene 5’-TAGAATTCATGATCGCCGCAAT-3’ 22-mer - encoding TES-32 TES-32R RT-PCR of ORF of gene 5’-GCATAAGCTTCTAGAGAGGTCTCTT-3’ 25-mer - encoding TES-32 TES-120F RT-PCR of ORF of 5’ – AGC CAC GCA ATG CAC GTC CTT ACC GTC 30-mer Fong et al., 2003 gene encoding TES-120 GCT – 3’ TES-120R RT-PCR of ORF of 5’ – CGA TCA TTA ACA GAA GCC GCA CGT CAG 30-mer Fong et al., 2003 gene encoding TES-120 TGG – 3’ M13F Analysis of recombinant 5’ – GTA AAA CGA CGG CCA G – 3’ 16-mer Instruction manual of TOPO TOPO-TA Cloning kit (Invitrogen, USA) M13R Analysis of recombinant 5’ – CAG GAA ACA GCT ATG AC – 3’ 17-mer Instruction manual of TOPO TOPO-TA Cloning kit (Invitrogen, USA) M13Rpuc Analysis of recombinant 5’ – AGC GGA TAA CAA TTT CAC ACA GG – 3’ 23-mer Instruction manual of pPROEXHT pPROEXHT Prokaryotic Expression System (Life Technologies, USA) T7 promoter Analysis of recombinant 5’ – TAA TAC GAC TCA CTA TAG GG – 3’ 20-mer Instruction manual of pET pET System Manual (Novagen, Germany)
103 Table 3.2 The expected PCR product size for ORF of each gene
Genes encoding Genbank Primers PCR product Accession no. size
TES-26 U29761 TES-26F (Tm 60.63, %GC 46.4) 793 bp TES-26R(Tm 60.84, %GC 50.0)
TES-32 AF041023 TES-32F (Tm 61.0, %GC 55.0) 660 bp TES-32R (Tm 60.5, %GC 58.0)
TES-120 U39815 TES-120F(Tm 64.5, %GC 57.1) 528 bp TES-120R (Tm 66.5, %GC 69.0)
3.2.3 Construction of TES-26, TES-32 and TES-120 recombinant plasmids
104 3.2.3.1 RT-PCR of ORF of the genes encoding TES-26, TES-32 and TES-120
RT-PCR was performed by using commercial “StrataScriptTM One-Tube RT-PCR System with Easy-ATM High-Fidelity PCR Cloning Enzyme” kit (Stratagene, USA) following the manufacturer’s instruction which combine cDNA synthesis, high-fidelity PCR amplification and the generation of 3’-A overhangs.
The experimental reaction comprised of 39.5 µl RNase-free water, 5 µl 10X RT-
PCR buffer, 1 µl of each forward and reverse primers (20 pmol/µl each), 1 µl 40 mM dNTP mix, 1 µl of 0.16 µg mRNA sample (1 µl of 1.6 µg/ µl mRNA sample was diluted with 9 µl of RNase-free water), 1 µl diluted StrataScript RT (2.5 U/µl) and 0.5 µl Easy-A HiFi PCR cloning enzyme. These were added sequentially in 0.2-ml PCR tube to make up a total reaction volume of 50 µl. A positive control reaction was run together using control primer set and control mRNA supplied in the kit. A negative PCR control was included in all the mixtures, except for the mRNA sample. The reaction mixture was gently vortexed. The amplification process was carried out using thermal cycler (MJ Research, Biorad) following the thermal cycling program as follows: first-strand synthesis at 42ºC for 15 minutes;
StrataScript RT inactivation at 95ºC for 1 minute; denaturation at 95ºC for 30 sec, template- primer annealing at 50ºC for 30 sec, extension at 68ºC for 2 minutes (40 cycles) and final extension at 68ºC for 5 minutes. The amplified RT-PCR product (50 µl) was analyzed by electrophoresis using horizontal 1% agarose gel, stained with ethidium bromide with a 100 bp ladder (Fermentas, USA) as the molecular weight marker (described in section 2.2.5) and readily visible by UV transilluminator.
The agarose gel analysis of RT-PCR products are shown in figures 3.2a – 3.2c. The targeted amplified bands of 793 bp (TES-26; figure 3.2a), 660 bp (TES-32; figure 3.2b) and
528 bp (TES-120; figure 3.2c) were detected (lane 1). The positive control reaction (lane 2), which contains the control mRNA and the control primer set, yields a 500-bp product. No band was observed in the negative PCR control lane (lane 3).
105 900 bp 800 bp 700 bp 600 bp 500 bp
M 1 2 3
Figure 3.2a RT-PCR product of ORF of the gene encoding TES-26 resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1 : RT-PCR product of ORF of the gene encoding TES-26 Lane 2 : Positive PCR control Lane 3 : Negative PCR control
106 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp
M 1 2 3
Figure 3.2b RT-PCR product of ORF of the gene encoding TES-32 resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1 : RT-PCR product of ORF of the gene encoding TES-32 Lane 2 : Positive PCR control Lane 3 : Negative PCR control
107 Figure 3.2c RT-PCR product of ORF of the gene encoding TES-120 resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1 : RT-PCR product of ORF of the gene encoding TES-120 Lane 2 : Positive PCR control Lane 3 : Negative PCR control
108 3.2.3.1.1 Purification of RT-PCR products
Prior to cloning, the RT-PCR products of TES-26, TES-32 and TES-120 were eluted out from the agarose gel and purified by using “Perfectprep Gel Cleanup” kit (Eppendorf, USA) as described in section 2.2.6. The purified products were then used for cloning into pCR 2.1
TOPO-TA cloning vector.
3.2.3.2 Cloning of RT-PCR products into pCR2.1 TOPO-TA vector
The A-tailed fresh purified RT-PCR products were cloned into pCR2.1-TOPO vector, supplied in the “TOPO-TA Cloning” kit (Invitrogen, USA). The features and sequence characteristics of pCR2.1-TOPO vector for the cloning regions is shown in figure 3.3. The reaction consisted of 2.5 µl of purified RT-PCR product, 1 µl of salt solution, 0.5 µl of pCR2.1-TOPO vector and 1 µl of water (DNAse & RNAse-free, Sigma, USA). The cloning reaction mix was gently mixed and incubated at room temperature for 5 minutes. The cloning reaction mix was then used to transform into E. coli TOP10 competent cell in blue- white selection of recombinant bacterial clones as described in section 2.2.8.
3.2.3.3 Verification of positive clones
3.2.3.3.1 PCR screening of TOPO/transformants
Ten white colonies from each of the TOPO/transformants (recombinant plasmids) were randomly selected and screened for inserts by colony PCR using a few set of primers: both
‘gene’-specific primers (TES-26F and TES-26R; TES-32F and TES-32R; TES-120F and
TES-120R); in tandem with vector-specific primer (external) and ‘gene’-specific primer
(internal) (M13R and TES-26R; M13R and TES-32R; M13R and TES-120R) in order to ascertain the orientation of the cloned fragment. PCR amplification was then carried out using the protocol described in section 2.2.4. After amplification, the PCR products were analyzed by using 1% horizontal DNA agarose gel electrophoresis (section 2.2.5).
109 Figure 3.3 Map and sequence characteristics of PCR2.1-TOPO vector shows the cloning region
* Sequence characteristics and map was adapted from the manufacturer (Invitrogen, USA).
110 Figures 3.4a-c shows the PCR results of TES-26, TES-32 and TES-120
TOPO/transformants, respectively. The results using both ‘gene’ specific primers showed that all ten white colonies (lane 1-10) showed the presence of amplified product bands corresponding to the expected size for TES-26, TES-32 and TES-120 (Set A, figures 3.4a-c).
Similarly, when using vector-specific primer (external) and ‘gene’-specific primer (internal), all ten colonies showed the presence of amplified products bands corresponding to the expected size for TES-26 and TES-32 (Set B, figures 3.4a & 3.4b). However, only six colonies gave the amplified band for TES-120 when using M13R and TES-120R (Set B, figure 3.4c). No band was observed in the negative PCR control (lane 11). Two positive clones from each recombinant plasmid with correct amplified product size were randomly selected and used for plasmid extraction (described in section 2.2.2).
3.2.3.3.2 Restriction endonuclease digestion of the recombinant plasmids
To verify the presence of ORF of the genes encoding TES-26, TES-32 and TES-120 in the constructed plasmids, a double restriction enzyme digestion was performed using BamH1 and Xho1 enzymes (Fermentas, USA). The digestion reaction consisted of the following: 2 μl of 10X enzyme buffer (BamH1 buffer), 3 μl of plasmid DNA (1μg), 1 μl of BamH1 and 2 μl of Xho1 enzyme (10 U/μl) each and sterile water to made up to a total volume of 20 μl.
Undigested plasmid DNA was included as a control of the test. The digestion reaction was mixed by gently tapping the bottom of the tube with fingers, then briefly centrifuged and incubated at 37ºC for 2 hours in a water bath. Immediately after the incubation time, the enzyme activity was stopped by incubating at 65ºC for 20 minutes followed by 1% DNA agarose gel analysis of the digested products as described earlier (subsection 2.2.5).
111 A
900 bp 800 bp 700 bp 793 bp 600 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
B 900 bp 800 bp 700 bp 883 bp 600 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
Figure 3.4a PCR product of TOPO/TES-26 transformants resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1-10: PCR products of transformants TES-26/TOPO/TOP10 Lane 11 : Negative PCR control
Set A: TES-26F and TES-26R primers [both ‘gene’-specific primers] Set B: M13R and TES-26R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
112
A 700 bp 600 bp 660 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
B
800 bp 700 bp 600 bp 750 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
Figure 3.4b PCR product of TOPO/TES-32 transformants resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1-10 : PCR products of transformants TES-32/TOPO/TOP10 Lane 11 : Negative PCR control
Set A : TES-32F and TES-32R primers [both ‘gene’-specific primers] Set B : M13R and TES-32R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
113 A
700 bp 600 bp 528 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
B 700 bp 660 bp 600 bp 500 bp
1 2 3 4 5 6 7 8 9 10 11 M
Figure 3.4c PCR screening of TOPO/TES-120 transformants resolved on 1% agarose electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1-10 : PCR products of transformants TOPO/TES-120 Lane 11 : Negative PCR control
Set A : TES-120F and TES-120R primers [both ‘gene’-specific primers] Set B : M13R and TES-120R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
114 Agarose gel analysis of the digested plasmids showed the presence of bands of approximately 870 bp (TES-26; figure 3.5a), 660 bp (TES-32; figure 3.5b) and 595 bp (TES-
120; figure 3.5c) corresponding to the expected size of the insert with part of the vector, and a higher band representing the linearized pCR2.1 TOPO vector of approximately size of
3700 bp. The presence of circular plasmid (3900 bp) was observed in the undigested plasmid reaction tubes.
3.2.3.3.3 Confirmation by DNA sequencing
The two clones from each recombinant plasmids (TOPO/TES-26; TOPO/TES-32;
TOPO/TES-120 #1 and #2) were sequenced using two vector-specific universal primers, which are the M13 Forward as the reverse primer and M13 Reverse as the forward primer to ensure that the gene of interest is in the correct orientation and unnecessary changes have not been introduced during the cloning steps. DNA sequencing was carried out by Solution for
Genetic Technology (SOLGENT), Korea.
The nucleotide sequences were analyzed using Vector NT1 version 6 software
(Invitrogen, USA). The contig alignment of the insert sequences were performed with the previously published sequence of the ORF of genes encoding TES-26, TES-32 or TES-120 from the Genbank. The sequencing results from each of the TOPO clones demonstrated the presence of ORF of genes encoding TES-26, TES-32 or TES-120. Sequences analysis revealed that the TES-32 and TES-120 plasmids corresponded (100% identity) to their reported sequences in the Genbank (figures 3.6b and 3.6c, respectively). The ORF of the genes of TES-26 and TES-32 were confirmed to be in the correct orientation, however, ORF of the genes encoding TES-120 was in the antisense orientation. However, this condition did not affect the subcloning into the expression vector since digestion with EcoR1 enzyme would generate a fragment with identical sticky ends and enable a good chance of obtaining recombinant plasmids in the sense orientation in the pPROExTMHT bacterial expression vector.
115 3900 bp 3700 bp
900 bp 1500 bp 800 bp 700 bp 1000 bp 600 bp 750 bp 500 bp 500 bp 400 bp 250 bp
M1 1 2 3 M2
Figure 3.5a Restriction enzyme analysis of the TOPO/TES-26 construct resolved on 1% agarose gel electrophoresis
Lane M1 : 100 bp ladder (Fermentas, USA) Lane M2 : 1 kb DNA ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-26 plasmid Lane 2 & 3 : Digested TOPO/TES-26 #1 and #2 plasmids
116 3900 bp 3700 bp
2000 bp 1500 bp
1000 bp 750 bp 500 bp
M 1 2 3
Figure 3.5b Restriction enzyme analysis of the TOPO/TES-32 constructs resolved on 1% agarose gel electrophoresis
Lane M : 1 kb DNA ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-32 plasmid Lane 2 & 3 : Digested TOPO/TES-32 #1 and #2 plasmids
117 3900 bp 3700 bp
750 bp
M 1 2 3
Figure 3.5c Restriction enzyme analysis of the TOPO/TES-120 constructs resolved on 1% agarose gel electrophoresis
Lane M : 1 kb DNA ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-120 plasmid Lane 2 & 3 : Digested TOPO/TES-120 #1 and #2 plasmids
118 Start codon
TES26 (1) GGTTTCATCATGTCAGTTGTACACAAAGCTTGCTTAAT TES26M13R67)TGCTGGAATTCGCCCTTCACCATGTCAGTTGTACACAAAGCTTGC TTAAT TES26M13F251TGCTGGAATTCGCCCTTCACCATGTCAGTTGTACACAAAGCTTGC TTAAT Consensus251TGCTGGAATTCGCCCTTCACCATGTCAGTTGTACACAAAGCTTGCTT AAT
TES26 (39)AGCTTTACTGTTTGTGTCGAGTGGAGTGGCACAACAGTGTATGGACAGTG TES26M13R117AGCTTTACTGTTTGTGTCGAGTGGAGTGGCACAACAGTGTATGG ACAGTG TES26M13F301AGCTTTACTGTTTGTGTCGAGTGGAGTGGCACAACAGTGTATGGA CAGTG Consensus301AGCTTTACTGTTTGTGTCGAGTGGAGTGGCACAACAGTGTATGGAC AGTG
TES26 (89)CGTCAGACTGTGCGGCAAACGCTGGGTCCTGTTTCACACGACCAGTCAGC TES26M13R167CGTCAGACTGTGCGGCAAACGCTGGGTCCTGTTTCACACGACCA ATCAGC TES26M13F351CGTCAGACTGTGCGGCAAACGCTGGGTCCTGTTTCACACGACCA ATCAGC Consensus351CGTCAGACTGTGCGGCAAACGCTGGGTCCTGTTTCACACGACCANT CAGC
TES26 (139)CAAGTTCTGCAGAACAGGTGTCAAAGGACATGCAATACATGCGACTGCCG TES26M13R217CAAGTTCTGCAGAACAGATGTCAAAGGACATGCAATACATGCGA CTGCCG TES26M13F401CAAGTTCTGCAGAACAGATGTCAAAGGACATGCAATACATGCGA CTGCCG Consensus401CAAGTTCTGCAGAACAGATGTCAAAGGACATGCAATACATGCGAC TGCCG
TES26 (189)GGACGAGGCTAACAATTGTGCAGCTTCAATTAACCTCTGCCAGAACCCCA TES26M13R267GGACGAGGCTAACAATTGTGCAGCTTCAATTAACCTCTGCCAGA ACCCCA TES26M13F451GGACGAGGCTAACAATTGTGCAGCTTCAATTAACCTCTGCCAGA ACCCCA Consensus451GGACGAGGCTAACAATTGTGCAGCTTCAATTAACCTCTGCCAGAAC CCCA
TES26 (239)CATTTGAACCTTTAGTTCGTGATCGATGCCAAAAGACTTGTGGTTTGTGT TES26M13R317CATTTGAACCTTTAGTTCGTGATCGATGCCAAAAGACTTGTGGTT TGTGT
119 TES26M13F501CATTTGAACCTTTAGTTCGTGATCGATGCCAAAAGACTTGTGGTT TGTGT Consensus501CATTTGAACCTTTAGTTCGTGATCGATGCCAAAAGACTTGTGGTTT GTGT
TES26 (289)GCGGGGTGCGGATTCATCAGTAGCGGAATCGTTCCATTAGTTGTGACGAG TES26M13R367GCGGGGTGCGGATTCATCAGTAGCGGAATCGTTCCATTAGTTGT GACGAG TES26M13F551GCGGGGTGCGGATTCATCAGTAGCGGAATCGTTCCATTAGTTGTG ACGAG Consensus551GCGGGGTGCGGATTCATCAGTAGCGGAATCGTTCCATTAGTTGTGA CGAG
TES26 (339)TGCACCTTCAAGACGCGTGAGTGTGACCTTCGCTAACAACGTTCAGGTGA TES26M13R417TGCACCTTCAAGACGCGTGAGTGTGACCTTCGCTAACAACGTTC AGGTGA TES26M13F601TGCACCTTCAAGACGCGTGAGTGTGACCTTCGCTAACAACGTTCA GGTGA Consensus601TGCACCTTCAAGACGCGTGAGTGTGACCTTCGCTAACAACGTTCAG GTGA
TES26 (389)ACTGCGGTAATACTTTGACGACGGCGCAAGTAGCCAACCAACCGACAGTA TES26M13R467ACTGCGGTAATACTTTGACGACGGCGCAAGTAGCCAACCAACCG ACAGTA TES26M13F651ACTGCGGTAATACTTTGACGACGGCGCAAGTAGCCAACCAACCG ACAGTA Consensus651ACTGCGGTAATACTTTGACGACGGCGCAAGTAGCCAACCAACCGA CAGTA
Figure 3.6a Contig alignment analysis of TOPO/TES-26#1 and previously published sequence of TES-26
The mutations bp is shown in box. Nucleic acid sequences in bold represent the start and stop codons.
TES26 (439)ACGTGGGAAGCGCAACCGAATGACAGGTACACCCTCATCATGGTCGATCC TES26M13R517ACGTGGGAAGCGCAACCGAATGACAGGTACACCCTTATCATGGT CGATCC TES26M13F701ACGTGGGAAGCGCAACCGAATGACAGGTACACCCTTATCATGGT CGATCC Consensus701ACGTGGGAAGCGCAACCGAATGACAGGTACACCCTTATCATGGTC GATCC
TES26 (489)TGACTTCCCAAGCGCGGCGAACGGCCAGCAGGGTCAGCGACTTCACTGGT TES26M13R567TGACTTCCCAAGCGCCGCGAACCGCCAGCAGGGTCAGCGACTTC ACTGGT
120 TES26M13F751TGACTTCCCAAGCGCCGCGAACCGCCAGCAGGGTCAGCGACTTC ACTGGT Consensus751TGACTTCCCAAGCGCCGCGAACNGCCAGCAGGGTCAGCGACTTCA CTGGT
TES26 (539)GGGTAATAAACATTCCAGGAAATAATATCGCCGGTGGTACGACGCTCGCA TES26M13R617GGGTAATAAACATTCCAGGAAATAATATCGCCGGTGGTACGACG CTCGCA TES26M13F801GGGTAATAAACATTCCAGGAAATAATATCGCCGGTGGTACGACG CTCGCA Consensus801GGGTAATAAACATTCCAGGAAATAATATCGCCGGTGGTACGACGC TCGCA
TES26 (589)GCGTTCCAGCCATCAACACCCGCCGCTAATACTGGTGTGCACCGATACGT TES26M13R667GCGTTCCAGCCATCAACACCCGCCGCTAATAGTGGTGGGCACCG ATACGT TES26M13F851GCGTTCCAGCCATCAACACCCGCCGCTAATAGTGGTGGGCACCG ATACGT Consensus851GCGTTCCAGCCATCAACACCCGCCGCTAATANTGGTGGGCACCGAT ACGT
TES26 (639)GTTTCTGGTATACAGACAGCCTGCCGCAATAAACTCTCCGCTACTTAATA TES26M13R717GTTTCTGGTATACAGACAGCCTGCCGCAATAAACTCTCCGCTACT TAATA TES26M13F901GTTTCTGGTATACAGACAGCCTGCCGCAATAAACTCTCCGCTACT TAATA Consensus901GTTTCTGGTATACAGACAGCCTGCCGCAATAAACTCTCCGCTACTT AATA
TES26 (689)ATTTGGTAGTGCAGGATTCGGAGCGGCCAGGTTTTGGTACGACTGCCTTT TES26M13R767ATTTGGTAGTGCAGGATTCGGAGCGGCCAGGTTTTGGTACGACT GCCTTT TES26M13F951ATTTGGTAGTGCAGGATTCGGAGCGGCCAGGTTTTGGTACGACT GCCTTT Consensus951ATTTGGTAGTGCAGGATTCGGAGCGGCCAGGTTTTGGTACGACTGC CTTT
TES26 (739)GCCACTCAGTTTAACCTTGGATCACCCTATGCTGGCAATTTCTATCGATC TES26M13R817GCCACTCAGTTTAACCTTGGATCACCCTATGCTGGCAACTTCTAT CGATC TES26M3F1001GCCACTCAGTTTAACCTTGGATCACCCTATGCTGGCAACTTCTAT CGATC Consens 1001GCCACTCAGTTTAACCTTGGATCACCCTATGCTGGCAANTTCTATCGATC Stop codon
TES26 (789)GCAGGCCTAATAGTCG-GTACTTCCGATGTGCA--A-A-TAAC-TCCG-- TES26M13R867GCAGGCCTAA- GGGCGAATTCTGCAGATATCCATCACACTGGCGGCCGCT
121 TES26M3F1051GCAGGCCTAA- GGGCGAATTCTGCAGATATCCATCACACTGGCGGCCGCT Consens1051)GCAGGCCTAANGGGCGAATTCTGCAGATATCCATCACACTGGCGGC CGCT
Figure 3.6a (cont.) Contig alignment analysis of TOPO/TES-26#1 and previously published sequence of TES-26
*The mutations bp is shown in box. Nucleic acid sequences in bold represent the start and stop codons.
Start codon
TES32(1) ATGATCGCCGCAATCGTCGTTTTGTTATG T32F(401) TGCTGGAATTCGCCCTTCAGGATGATCGCCGCAATCGTCGTTTTGTTATG T32R(66) TGCTGGAATTCGCCCTTCAGGATGATCGCCGCAATCGTCGTTTTGTTATG Consens401TGCTGGAATTCGCCCTTCAGGATGATCGCCGCAATCGTCGTTTTGTTA TG
TES32(30) TCTCCACCTGTCCACCACAAATGCTTGCGCCACCAACAATGACTGTGGTA T32F(451) TCTCCACCTGTCCACCACAAATGCTTGCGCCACCAACAATGACTGTGGTA T32R(116) TCTCCACCTGTCCACCACAAATGCTTGCGCCACCAACAATGACTGTGGTA Consen451 TCTCCACCTGTCCACCACAAATGCTTGCGCCACCAACAATGACTGTGGTA
TES32(80) TTTTCCAAGTGTGCGTTAACAACGTCTGCGTTGCCAATAACCAAGGATGC T32F(501) TTTTCCAAGTGTGCGTTAACAACGTCTGCGTTGCCAATAACCAAGGATGC T32R(166) TTTTCCAAGTGTGCGTTAACAACGTCTGCGTTGCCAATAACCAAGGATGC Consen501 TTTTCCAAGTGTGCGTTAACAACGTCTGCGTTGCCAATAACCAAGGATGC
TES32130) AATCCGCCTTGTGTGGCTCCGCAGGTTTGCGTTGCACCAATGTGTGTCGC T32F(551) AATCCGCCTTGTGTGGCTCCGCAGGTTTGCGTTGCACCAATGTGTGTCGC T32R(216) AATCCGCCTTGTGTGGCTCCGCAGGTTTGCGTTGCACCAATGTGTGTCGC Consen551 AATCCGCCTTGTGTGGCTCCGCAGGTTTGCGTTGCACCAATGTGTGTCGC
TES32180) CCCTCCACCGGCAGCCACTACAACTGCAGCTCCAGGCGTTACAACTACAA
122 T32F 601) CCCTCCACCGGCAGCCACTACAACTGCAGCTCCAGGCGTTACAACTACAA T32R 266) CCCTCCACCGGCAGCCACTACAACTGCAGCTCCAGGCGTTACAACTACAA Consen601 CCCTCCACCGGCAGCCACTACAACTGCAGCTCCAGGCGTTACAACTACAA
TES32230) GACCACGTGCCTGCCCACCCAATTGGACGCCGTTCAACAACAATTGCTAT T32F 651) GACCACGTGCCTGCCCACCCAATTGGACGCCGTTCAACAACAATTGCTAT T32R 316) GACCACGTGCCTGCCCACCCAATTGGACGCCGTTCAACAACAATTGCTAT Consen651)GACCACGTGCCTGCCCACCCAATTGGACGCCGTTCAACAACAATTGC TAT
TES32280) ATCGCCTCTCTACCTGGACGCTTCCTTTTCAACCAAGCTAGCGACTGGTG T32F(701) ATCGCCTCTCTACCTGGACGCTTCCTTTTCAACCAAGCTAGCGACTGGTG T32R(366) ATCGCCTCTCTACCTGGACGCTTCCTTTTCAACCAAGCTAGCGACTGGTG Consen701 ATCGCCTCTCTACCTGGACGCTTCCTTTTCAACCAAGCTAGCGACTGGTG
TES32330) TACACAGACTGGCTCAAGAGTTGTGTGGTTCGACCAGTCGACTGTAGGAA T32F(751) TACACAGACTGGCTCAAGAGTTGTGTGGTTCGACCAGTCGACTGTAGGAA T32R(416) TACACAGACTGGCTCAAGAGTTGTGTGGTTCGACCAGTCGACTGTAGGAA Consen751)TACACAGACTGGCTCAAGAGTTGTGTGGTTCGACCAGTCGACTGTAG GAA
Figure 3.6b Contig alignment analysis of TOPO/TES-32 and previously published sequence of TES-32
* Nucleic acid sequences in bold represent the start and stop codons.
TES32 380) ACTTCGGCAGCGAACTGAACTTCGTCAACAGTTTCGCTGTTGGTCGTGGA T32F (801) ACTTCGGCAGCGAACTGAACTTCGTCAACAGTTTCGCTGTTGGTCGTGGA T32R (466) ACTTCGGCAGCGAACTGAACTTCGTCAACAGTTTCGCTGTTGGTCGTGGA
123 Consen801) ACTTCGGCAGCGAACTGAACTTCGTCAACAGTTTCGCTGTTGGTCGTGGA
TES32(430) GTAACCAGGTACTGGATAGGAGTGAACAGACAGTTCGGCCAGTGGGTGTT T32F (851) GTAACCAGGTACTGGATAGGAGTGAACAGACAGTTCGGCCAGTGGGTGTT T32R (516) GTAACCAGGTACTGGATAGGAGTGAACAGACAGTTCGGCCAGTGGGTGTT Consen851) GTAACCAGGTACTGGATAGGAGTGAACAGACAGTTCGGCCAGTGGGTGTT
TES32(480) TACGAATGGAAGTCCAGTGATATTTTCGAACTGGAGGCCTAGTCAACCGG T32F (901) TACGAATGGAAGTCCAGTGATATTTTCGAACTGGAGGCCTAGTCAACCGG T32R (566) TACGAATGGAAGTCCAGTGATATTTTCGAACTGGAGGCCTAGTCAACCGG Consen901) TACGAATGGAAGTCCAGTGATATTTTCGAACTGGAGGCCTAGTCAACCGG
TES32(530) ACGGATGCTGTGGTTCCAACGTCACGTGTGCGTTCGTTAATTACGCGAAC T32F (951) ACGGATGCTGTGGTTCCAACGTCACGTGTGCGTTCGTTAATTACGCGAAC T32R (616) ACGGATGCTGTGGTTCCAACGTCACGTGTGCGTTCGTTAATTACGCGAAC Consen951) ACGGATGCTGTGGTTCCAACGTCACGTGTGCGTTCGTTAATTACGCGAAC
TES32(580) TTCCTCGGCCAATGGGATGATGCCCCATGCGGAAGTTTGTTTACGACTCC T32F(1001) TTCCTCGGCCAATGGGATGATGCCCCATGCGGAAGTTTGTTTACGACTCC T32R (666) TTCCTCGGCCAATGGGATGATGCCCCATGCGGAAGTTTGTTTACGACTCC Consen1001 TTCCTCGGCCAATGGGATGATGCCCCATGCGGAAGTTTGTTTACGACTCC
Stop codon
TES32 630) ACAAGGCTTCGTATGCAAGAGACCTCTCTAG T32F 1051) ACAAGGCTTCGTATGCAAGAGACCTCTCTAGATTCAAGGGCGAATTCTGC T32R (716) ACAAGGCTTCGTATGCAAGAGACCTCTCTAGATTCAAGGGCGAATTCTGC Consen1051)ACAAGGCTTCGTATGCAAGAGACCTCTCTAGATTCAAGGGCGAATT CTGC
124 Figure 3.6b (cont.) Contig alignment analysis of TOPO/TES-32 and previously published sequence of TES-32
* Nucleic acid sequences in bold represent the start and stop codons.
Start codon
TES120F601) TGATGGAT-ATCTGCAGAA-TTCG-----CC-CTTAATGCACGTCCTTAC n-muc1 (1) GGTTCAATTACCCAAGTTGGAGGAGCCACGCAATGCACGTCCTTAC TES120R86) TGATGGAT-ATCTGCAGAA-TTCG-----CC-CTTAATGCACGTCCTTAC Consens 601TGATGGATNATCTGCAGAANTTCGNNNNNCCNCTTAATGCACGTCCTTAC
TES120F 651)CGTCGCTCTCGTCGCAGTGCTAATCTGCGTTGCTACACCACAAATGATGT n-muc1 (47) CGTCGCTCTCGTCGCAGTGCTAATCTGCGTTGCTACACCACAAATGATGT TES120R 136)CGTCGCTCTCGTCGCAGTGCTAATCTGCGTTGCTACACCACAAATGATGT Consens 651CGTCGCTCTCGTCGCAGTGCTAATCTGCGTTGCTACACCACAAATGATGT
TES120F701 CCAGTTCCTCTTCATCGTCTCCGTCAACATCCTCATCGTCAGCGTCAACA n- muc1 (97) CCAGTTCCTCTTCATCGTCTCCGTCAACATCCTCATCGTCAGCGTCAACA TES120R186 CCAGTTCCTCTTCATCGTCTCCGTCAACATCCTCATCGTCAGCGTCAACA Consens701) CCAGTTCCTCTTCATCGTCTCCGTCAACATCCTCATCGTCAGCGTCAACA
TES120F 751)TCCTCATCGTCCGCGTCAACATCCTCATCGTCCGCGTCAACATCCTCATC n-muc1(147) TCCTCATCGTCCGCGTCAACATCCTCATCGTCCGCGTCAACATCCTCATC TES120R(236)TCCTCATCGTCCGCGTCAACATCCTCATCGTCCGCGTCAACATCCT CATC Consens(751)TCCTCATCGTCCGCGTCAACATCCTCATCGTCCGCGTCAACATCCTC ATC
TES120F(801)GTCCGCGTCAACATCCTCATCGCCCGCGTCAACATCCTCATCGTCC GCGT n-muc1(197) GTCCGCGTCAACATCCTCATCGCCCGCGTCAACATCCTCATCGTCCGCGT TES120R(286)GTCCGCGTCAACATCCTCATCGCCCGCGTCAACATCCTCATCGTCC GCGT Consens(801)GTCCGCGTCAACATCCTCATCGCCCGCGTCAACATCCTCATCGTCCG CGT
125 TES120F(851)CAACAAGCTCAATGGCAGGATCTACATCCACAGCAGCTGGGCCGA CATCC n-muc1(247) CAACAAGCTCAATGGCAGGATCTACATCCACAGCAGCTGGGCCGACATCC TES120R(336)CAACAAGCTCAATGGCAGGATCTACATCCACAGCAGCTGGGCCGA CATCC Consens(851)CAACAAGCTCAATGGCAGGATCTACATCCACAGCAGCTGGGCCGAC ATCC
TES120F(901)AGCTCTTCTGTTTCCACATCAACTCCAGCGGTGATGACAACGACGC CAGC n-muc1(297) AGCTCTTCTGTTTCCACATCAACTCCAGCGGTGATGACAACGACGCCAGC TES120R(386)AGCTCTTCTGTTTCCACATCAACTCCAGCGGTGATGACAACGACGC CAGC Consens(901)AGCTCTTCTGTTTCCACATCAACTCCAGCGGTGATGACAACGACGC CAGC
TES120F(951)GTGCATCGACACCGCTAACGACTGCCAGCTGTTCACGCCACTCTGC TTCG n-muc1(347) GTGCATCGACACCGCTAACGACTGCCAGCTGTTCACGCCACTCTGCTTCG TES120R(436)GTGCATCGACACCGCTAACGACTGCCAGCTGTTCACGCCACTCTGC TTCG Consens(951)GTGCATCGACACCGCTAACGACTGCCAGCTGTTCACGCCACTCTGC TTCG
TES120F(1001TTCAGCCATACAGCAGAGCAATACAGGGAAGATGCCGAAGGACG TGCAAC n-muc1(397) TTCAGCCATACAGCAGAGCAATACAGGGAAGATGCCGAAGGACGTGCAAC TES120R(486)TTCAGCCATACAGCAGAGCAATACAGGGAAGATGCCGAAGGACG TGCAAC Consen(1001)TTCAGCCATACAGCAGAGCAATACAGGGAAGATGCCGAAGGACGT GCAAC
Figure 3.6c Contig alignment analysis of TOPO/TES-120 and previously published sequence of TES-120
* Nucleic acid sequences in bold represent the start and stop codons.
TES120F1051)ATTTGCAGCTGTCAGGACAGTGCCAACGACTGTGCAAACT TTGTTTCAGT n-muc1(447) ATTTGCAGCTGTCAGGACAGTGCCAACGACTGTGCAAACTTTGTTTCAGT TES120R(536)ATTTGCAGCTGTCAGGACAGTGCCAACGACTGTGCAAACTTTGTTT CAGT Consen(1051)ATTTGCAGCTGTCAGGACAGTGCCAACGACTGTGCAAACTTTGTTT CAGT
TES120F1101)CTGCCTGAACCCGACCTATCAGCCAGTGCTTCGATCAAGATGCCC ACTGA n-muc1(497) CTGCCTGAACCCGACCTATCAGCCAGTGCTTCGATCAAGATGCCCACTGA TES120R(586)CTGCCTGAACCCGACCTATCAGCCAGTGCTTCGATCAAGATGCCC ACTGA
126 Consen(1101)CTGCCTGAACCCGACCTATCAGCCAGTGCTTCGATCAAGATGCCCA CTGA
Stop codon
TES120F1151)CGTGCGGCTTCTGTTAAGGG-CGA-A-T-TC-C-A-GCAC--ACTGGCGG n-muc1(547) CGTGCGGCTTCTGTTAATGATCGGTAGTATCGCGACGAACTGAATGGCTT TES120R(636)CGTGCGGCTTCTGTTAAGGG-CGA-A-T-TC-C-A-GCAC--ACTGGCGG Consen(1151)CGTGCGGCTTCTGTTAAGGGNCGANANTNTCNCNANGCACNNACTG GCGG
Figure 3.6c (cont.) Contig alignment analysis of TOPO/TES-120 and previously published sequence of TES-120
* Nucleic acid sequences in bold represent the start and stop codons.
127 However, sequence analysis of the two TOPO/TES-26 plasmids revealed four mutations (at the same sites in both plasmids) with respect to the published TES-26 sequence
(figure 3.6a). The mutations occured at the following base positions: 124 bp (G replaced by
A); 502 bp (G replaced by C); 613 bp (C replaced by G); 768 (T replaced by C). These mutations resulted in changes of amino acid and probably occurred during the PCR syntesis process.
3.2.3.4 In-vitro mutagenesis
In-vitro site directed mutagenesis was performed to correct the base-errors in TOPO/TES-
26#1 clone following the guidelines described by manufacturer (Stratagene, USA). The PCR was performed using Quickchange XL Site-directed mutagenesis kit (Stratagene, USA).
Briefly, PCR-based site-directed mutagenesis was performed using PfuTurbo® DNA polymerase which replicates both plasmid strands with high fidelity and without displacing the mutant oligonucleotide primers. Overview of the Quickchange® site-directed mutagenesis process is shown in Figure 3.7.
3.2.3.4.1 PCR-based site-directed mutagenesis
The mutagenic primers (listed in Table 3.3) were designed following the guidelines described by manufacturer (Stratagene, USA). Four microliter of plasmid DNA template was added into a PCR reaction mixture consisting of 5 µl of 10X mutagenesis buffer, 2 µl of each
1 µM of forward and reverse mutagenic primers, 1 µl of 10 mM dNTPs, 1 µl of Pfu DNA polymerase (2.5U/µl) and water (DNAse-RNAse-free, Sigma, USA) to a final volume of
50 µl. PCR was performed using the following parameters: 1 cycle at 95ºC (5 minutes); 12 cycles at 95ºC (30 sec), 55/60ºC (1 minute) and 72ºC (5 minutes) and finally 1 cycle at 4ºC
(5 minutes).
128 After amplification process, the PCR products were kept on ice for 2 minutes and 10
µl of amplified products were analyzed by 1% DNA agarose gel electrophoresis as described
in section 2.2.5. Figure 3.8 showed the presence of two bands (lane 1); the upper band was
Step 1 Plasmid Preparation Gene in plasmid with target site for mutation
Step 2 Temperature Cycling Denaturation of the plasmid and annealing of oligonucleotide primers containing the desired mutation Mutagenic primers
Using the nonstrand-displacing action of PfuTurbo® DNA polymerase, the mutagenic primers were extended and incorporated resulted in nicked circular strands
Step 3 Digestion Methylated, nonmutated parental DNA template was digested with Dpn1 Mutated plasmid (contain nicked circular strand) Step 4 Circular, nicked dsDNA was Transformation transformed into E.coli competent cells
After transformation, the E.coli competent cells repaired the nicks in the mutated plasmid
Figure 3.7 Schematic overview of the Quickchange® site-directed mutagenesis method (Stratagene, USA) ( Parental DNA plasmid, Mutagenic primer, Mutated DNA plasmid) [adapted from the manufacturer (Stratagene, USA)].
129 Table 3.3 Primers used for repairing base-errors in site-directed mutagenesis
Mutagenic primers Sequence (5’ – 3’) (25-mer) Purpose
TES26MF1 ttcacacgaccagtcagccaagttc G to replace A
TES26MR1 gaacttggctgactggtcgtgtgaa G to replace A
TES26MF2 agcgccgcgaacggccagcagggtc G to replace C
TES26MR2 gaccctgctggccgttcgcggcgct G to replace C
TES26MF3 ccgccgctaatactggtgtgcaccg C to replace G
TES26MR3 cggtgcacaccagtattagcggcgg C to replace G
TES26MF4 tatgctggcaatttctatcgatcgc T to replace C
TES26MR4 gcgatcgatagaaattgccagcata T to replace C
130 Set A Set B
1500 bp 900 bp 1500 bp 800 bp 1000 bp 800 bp 1000 bp 700 bp 750 bp 700 bp 750 bp 600 bp 600 bp 500 bp 500 bp 500 bp 500 bp 400 bp 400 bp
M2 1 2 M1 M2 1 2 M1
Set C Set D
900 bp 1500 bp 800 bp 1500 bp 800 bp 1000 bp 700 bp 1000 bp 700 bp 750 bp 600 bp 750 bp 600 bp 500 bp 500 bp 500 bp 400 bp 500 bp 400 bp
M2 1 2 M1 M2 1 2 M1
Figure 3.8 Agarose gel electrophoresis analysis of the site-directed mutagenesis product of TOPO/TES26
Lane M1 : 100 bp DNA ladder (Fermentas, USA) Lane M2 : 1 kb DNA ladder (Fermentas, USA) Lane 1 : Reaction mix (undigested) Lane 2 : Reaction mix after digestion with Dpn1.
Set A : Reaction mix to repair the first base error at position 124 bp Set B : Reaction mix to repair the second base error at position 502 bp Set C : Reaction mix to repair the third base error at position 613 bp Set D : Reaction mix to repair the fourth base error at position 768 bp
131 the newly synthesized DNA and the lower band was the parental plasmid. Subsequently, 1 µl of Dpn1 enzyme (10U/µl) was added into the remaining 40 µl amplified product and the mixture was incubated at 37ºC for 1 hour to digest the parental DNA plasmid. The reaction was then analyzed by 1% DNA agarose gel electrophoresis to confirm the digestion of parental plasmid. The nonmutated parental plasmids were successfully digested as shown by the presence of a lower band (lane 2).
Two µl of site-directed mutagenesis reaction mixture was then used to transform into
E. coli TOP10 competent cells as described in section 2.2.8. The plasmids DNA were extracted (section 2.2.2) from the TOPO/TES-26 transformants. These plasmids were then used in site-directed mutagenesis to repair the second base-error. The plasmids DNA were again extracted (section 2.2.2) from the second repair of TOPO/TES-26 transformants which were then used in a third mutagenesis experiment. The plasmids DNA were again extracted
(section 2.2.2) from the third repair of TOPO/TES-26 transformants and finally used in a fourth mutagenesis experiment. After all the base-errors were repaired, two final repaired clones (TOPO/TES-26#1c and #1d) were chosen for plasmid extraction followed by sending for sequencing to determine the success of the site-directed mutagenesis experiments. The sequencing results were analyzed as previously described (section 3.2.3.3.3). Three attempts had to be carried out to perform site-directed mutagenesis for the third mutation site until it was finally corrected.
Sequences analysis verified that one base error (at 613 bp) in TOPO/TES-26#1c was not repaired; however, all base errors in TOPO/TES-26#1d fragment were successfully repaired and were chosen for the subsequent experiment. The TOPO/TES-26#1d sequence was then compared with the pET42 expression vector using vector NT1 software to check for version suitability that can be used for expression. The TOPO/TES-26#1d plasmid was digested with EcoR1 enzyme and expressed in correct version of the pET42 expression vector so that it will be in the correct reading frame with the fusion tag and stop codon.
3.2.3.5 Cloning into expression vectors
132 The ORF of the genes encoding TES-26 and TES-32 were cloned into the pET-42 version
“b” and “a”, respectively, while the ORF of the gene encoding TES-120 was subcloned into the pPROExTMHT vectors. The features and sequence characteristics of pET-42 and pPROExTMHT vectors for the cloning and expression regions are shown in figures 3.9 and
3.10, respectively.
3.2.3.5.1 Vector and insert preparation
3.2.3.5.1.1 Single digestion with restriction enzyme
The plasmids DNA (inserts and vectors) were digested with the appropriate restriction endonuclease to preserve the reading frame of the protein as described in the manufacturer’s instructions. A single restriction enzyme digestion was performed on the extracted plasmids of TOPO/TES-26, TES-32 and TES-120 as well as pET-42 version “a”, “b” and pPROExTMHT version “a” using EcoR1 enzyme (Fermentas, USA) to generate fragment with EcoR1 digestion site flanking both sites of the fragment. The digestion reaction consisted of the following: 2 μl of 10X enzyme buffer (EcoR1 buffer), 3 μl of plasmid DNA
(1μg), 1 μl (1 Unit) of EcoR1 restriction enzyme and 14 μl of sterile water to make up a total volume of 20 μl. The digestion reaction was mixed by gently tapping the bottom of the tube with fingers, briefly centrifuged and incubated at 37ºC for 2 hours in a water bath.
Immediately after the incubation time, the enzyme activity was stopped by incubating at
65ºC for 20 minutes followed by 1% DNA agarose gel analysis of the digested products as described earlier (section 2.2.5).
Agarose gel analysis of the digested TOPO/TES-26, TES-32 and TES-120 showed the presence of a lower band of approximately 810 bp (TES-26, figure 3.11a), 700 bp (TES-
32, figure 3.11b) and 545 bp (TES-120, figure 3.11c), corresponding to the expected size, and a higher band representing the linearized PCR2.1 TOPO vector of approximately size of
3700 bp (lane 2) (figure 3.11). The presence of circular plasmid (3900 bp) was observed in
133 Figure 3.9a Map of pET-42a-c vector shows the multiple cloning regions
* Sequence characteristics and map was adapted from the manufacturer (Novagen, Germany).
134 Figure 3.9b Sequence characteristics of pET-42a-c vector shows the cloning and expression regions
* Sequence characteristics and map was adapted from the manufacturer (Novagen, Germany).
135 Figure 3.10 Map and multiple cloning sites of pPROExTMHT expression vectors
136 The schematic of pPROExTMHTa (4750 bp) with selected single digestion restriction endonucleases is presented. The sequence for the (his)6, spacer region and recombinant TEV protease cleavage site are underlined. The stop codon for each vector is underlined and italicized.
* Sequence characteristics and map was adapted from the manufacturer (Life Technologies, USA)
137 810 bp 1500 bp 800 bp 1000 bp 700 bp 750 bp 600 bp 500 bp 500 bp 250 bp
` M1 1 2 3 4 M2
Figure 3.11a Restriction enzyme analysis of TOPO/TES-26 and pET42b resolved on 1% DNA agarose gel electrophoresis
Lane M1 : 100 bp ladder (Fermentas, USA) Lane M2 : 1 kb DNA ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-26 Lane 2 : EcoR1 digested TOPO/TES-26 Lane 3 : Undigested pET42b Lane 4 : EcoR1 digested pET42b
138 2000 bp 700 bp 1500 bp
1000 bp 750 bp 700 bp 500 bp 600 bp 500 bp
M1 1 2 3 4 M2
Figure 3.11b Restriction enzyme analysis of TOPO/TES-32 and pET42a resolved on 1% DNA agarose gel electrophoresis
Lane M1 : 1 kb DNA ladder (Fermentas, USA) Lane M2 : 100 bp ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-32 Lane 2 : EcoR1 digested TOPO/TES-32 Lane 3 : Undigested pET42a Lane 4 : EcoR1 digested pET42a
139 800 bp 700 bp 600 bp 500 bp
M 1 2 3 4
Figure 3.11c Restriction enzyme analysis of TOPO/TES-120 and pPROExHTa resolved on 1% DNA agarose gel electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1 : Undigested TOPO/TES-120 Lane 2 : EcoR1 digested TOPO/TES-120 Lane 3 : Undigested pPROExTMHTa Lane 4 : EcoR1 digested pPROExTMHTa
140 the undigested plasmid (lane 1). The linearized pET42 and pPROExTMHT vectors (lane 4) size was lower than circularized plasmid vectors (lane 3).
3.2.3.5.1.2 Dephosphorylation of vectors
Since cloning was performed into a single site, the pET42 and pPROExTMHT vectors were dephosphorylated to decrease the background of non-recombinants due to self-ligation of the vector. Each of the digested pET42 “b” and “a” as well as pPROExTMHT “a” vectors were dephosphorylated by shrimp alkaline phosphatase (SAP) as follows: 20 μl of digested vector,
2 μl of 10X SAP buffer and 2 μl of SAP (Fermentas, USA) were mixed and the mixture was incubated at 37ºC for 30 minutes and the reaction was stopped by incubating the tube at 65ºC for 20 minutes.
3.2.3.5.1.3 Gel-purification of DNA inserts
Prior to cloning into pET42 and pPROExTMHT vectors, the EcoR1-digested product of TES-
26, TES-32 and TES-120 were gel-purified prior to ligation by using Perfectprep Gel
Cleanup kit (Eppendorf, USA) as described in section 2.2.6 to remove residual nicked and supercoiled plasmid, which transform very efficiently relative to the desired ligation products. The purified inserts (TES-26, TES-32 and TES-120) were then ready to be ligated to the pET42b, pET42a and pPROExTMHTa expression vectors, respectively.
3.2.3.5.2 Ligation of inserts into expression vectors
The ligation was performed by using Rapid Ligation Kit following the guidelines described by manufacturer (Roche Diagnostics, Germany). The ligation reaction of 21 μl consisted of 7
μl of gel-purified inserts, 2 μl of pPROExTMHT or pET42 vector, 1 μl of DNA dilution buffer and 10 μl of rapid ligation buffer. Reaction was started by adding 1 μl of T4 DNA ligase, mixed by pipetting and incubated at room temperature for 15 minutes. The circular recombinant plasmid is now ready for transformation into non-expression host as described
141 in section 2.2.8. There are no blue/white screening capabilities in the pET system because the pET vectors do not encode the lacZα-peptide.
3.2.3.5.3 Analysis of pPROExTMHT/ pET recombinants
3.2.3.5.3.1 PCR screening
Ten colonies of pPROExTMHT and pET transformants (recombinant plasmids) were randomly selected and screened for inserts by colony PCR using a few set of primers: both
‘gene’-specific primers; vector-specific primer (external) and ‘gene’-specific primers
(internal) (T7 promoter and reverse primer) in order to ascertain the orientation of the cloned fragment. PCR amplification was then carried out using the protocol described in section
2.2.4. The PCR products were analyzed by using 1% DNA agarose gel electrophoresis
(Section 2.2.5).
The PCR results showed that all selected colonies gave the expected bands of 793 bp for TES-26F and TES-26R primers (Set A) (Figure 3.12a). Similar to the PCR results of
TES-32 which showed all selected colonies gave the expected bands of 660 bp for TES-32F and TES-32R primers (Set A) (Figure 3.12b). However, only 8 out of 10 selected colonies gave the expected bands of 528 bp for TES-120F and TES-120R primers (Set A) (Figure
3.12c). The PCR screening using vector-specific and ‘gene’-specific primers showed that only 5 out of 10 selected colonies gave the expected size of approximately 1659 bp for T7 promoter and TES-26R primers (Set B) (Figure 3.12a). For TES-32, 8 out of 10 selected colonies gave the expected size of approximately 1530 bp using T7 promoter and TES-32R primers (Set B) (Figure 3.12b) while only 2 selected colonies from TES-120 transformants produced the expected band of approximately 624 bp for M13Rvspuc and TES-120R primers (Set B) (Figure 3.12c). Two positive clones from each recombinant gene with correct amplified product size were selected and used for plasmid extraction (described in section 2.2.2).
142 A
800 bp 700 bp 600 bp 500 bp 400 bp
M1 1 2 3 4 5 6 7 8 9 10 11
B 2000 bp 1500 bp 1000 bp 500 bp
1 2 3 4 5 6 7 8 9 10 11 M2
Figure 3.12a PCR screening of pET/TES-26 recombinants resolved on 1% agarose electrophoresis
Lane M1 : 100 bp ladder (Fermentas, USA) Lane M2 : 1 kb DNA ladder (Fermentas, USA) Lane 1-10 : PCR products of transformants TES-26/pET/TOP10 Lane 11 : Negative PCR control
Set A : TES-26F and TES-26R primers [both ‘gene’-specific primers] Set B : T7 promoter and TES-26R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
143 A
750 bp 500 bp
M 1 2 3 4 5 6 7 8 9 10 11
B
2000 bp 1500 bp 1000 bp
M 1 2 3 4 5 6 7 8 9 10 11
Figure 3.12b PCR screening of pET/32 recombinants resolved on 1% agarose electrophoresis
Lane M : 1 kb DNA ladder (Fermentas, USA) Lane 1-10 : PCR products of transformants TES-32/pET/TOP10 Lane 11 : Negative PCR control
Set A: TES-32F and TES-32R primers [both ‘gene’-specific primers] Set B: T7 promoter and TES-32R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
144 A 700 bp 600 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
B 800 bp 700 bp 600 bp 500 bp 400 bp
M 1 2 3 4 5 6 7 8 9 10 11
Figure 3.12c PCR screening of pPROExTMHT recombinants resolved on 1% agarose electrophoresis
Lane M : 100 bp ladder (Fermentas, USA) Lane 1-10 : PCR products of transformants TES-120/ pPROExTMHTa/ TOP10 Lane 11 : Negative PCR control
Set A : TES-120F and TES-120R primers [both ‘gene’-specific primers] Set B : M13Rvspuc and TES-120R primers [vector-specific primer (external) and ‘gene’-specific primer (internal)]
145 3.2.3.5.3.2 DNA sequencing
The two clones from each recombinant plasmids (designated as pET/TES-26#2 and #8; pET/TES-32#2 & #3; pPROExTMHT/TES-120#4 and #10) were sequenced using vector- specific and ‘gene’-specific primers to ensure that the gene of interest is in the correct reading frame. DNA sequencing was carried out by Solution for Genetic Technology
(SOLGENT), Korea.
The nucleotide sequences were translated into acid amino sequences and analyzed using Vector NT1 version 6 software (Invitrogen, USA). The contig alignments of the sequences of recombinant plasmids were performed with the previously published sequences of the genes encoding TES-26, TES-32 and TES-120 from the Genbank (Figure 3.13). All plasmid sequencing results revealed the presence of the genes encoding TES-26, TES-32 and
TES-120 in the correct reading frame inside the pET42b, pET42a and pPROExTMHTa expression vectors, respectively with appropriate fusion tags and stop codon. The sequences verified that the target genes were cloned correctly into an appropriate expression vector.
The pET42/TES-26#8, pET42/TES-32#2 and pPROExTMHT/TES-120#15 plasmids were then chosen for subsequent experiment.
3.2.3.5.4 Transformation of pPROExTMHT/pET recombinants into expression host
After the constructs were verified, plasmids were then transformed into BL21(DE3) as described in section 2.2.9 for protein production.
146 Start codon of vector
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val 1 ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG
Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys 43 CAA CCC ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys 82 TAT GAA GAG CAT TTG TAT GAG CGC GAT GAA GGT GAT AAA
Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro 122 TGG CGA AAC AAA AAG TTT GAA TTG GGT TTG GAG TTT CCC
Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln 161 AAT CTT CCT TAT TAT ATT GAT GGT GAT GTT AAA TTA ACA CAG
Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 205 TCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser 243 ATG TTG GGT GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA
Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val 283 ATG CTT GAA GGA GCG GTT TTG GAT ATT AGA TAC GGT GTT
Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys 323 TCG AGA ATT GCA TAT AGT AAA GAC TTT GAA ACT CTC AAA
Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met 361 GTT GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG AAA ATG
Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly 400 TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala 440 GAT CAT GTA ACC CAT CCT GAC TTC ATG TTG TAT GAC GCT
Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp 480 CTT GAT GTT GTT TTA TAC ATG GAC CCA ATG TGC CTG GAT
Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu 519 GCG TTC CCA AAA TTA GTT TGT TTT AAA AAA CGT ATT GAA
Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys 558 GCT ATC CCA CAA ATT GAT AAG TAC TTG AAA TCC AGC AAG
Figure 3.13a Amino acid sequence of pET42/TES-26 construct
* Nucleic acid sequences in bold represent the GST-tag.
Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe
147 597 TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC ACG TTT
Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr 636 GGT GGT GGC GAC CAT CCT CCA AAA TCG GAT GGT TCA ACT
His-tag
Ser Gly Ser Gly His His His His His His Ser Ala Gly Leu 678 AGT GGT TCT GGT CAT CAC CAT CAC CAT CAC TCC GCG GGT CTG
Val Pro Arg Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala 720 GTG CCA CGC GGT AGT ACT GCA ATT GGT ATG AAA GAA ACC GCT
Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro Asp Leu 762 GCT GCT AAA TTC GAA CGC CAG CAC ATG GAC AGC CCA GAT CTG
Gly Thr Gly Gly Gly Ser Gly Ile Glu Gly Arg Gly Ser Met 804 GGT ACC GGT GGT GGC TCC GGT ATT GAG GGA CGC GGG TCC ATG
Start codon of gene
Asp Ile Gly Asp Pro Asn Ser Pro Phe Thr Met Ser Val Val 846 GAT ATC GGG GAT CCG AAT TCG CCC TTC ACC ATG TCA GTT GTA
His Lys Ala Cys Leu Ile Ala Leu Leu Phe Val Ser Ser Gly 888 CAC AAA GCT TGC TTA ATA GCT TTA CTG TTT GTG TCG AGT GGA
Val Ala Gln Gln Cys Met Asp Ser Ala Ser Asp Cys Ala Ala 930 GTG GCA CAA CAG TGT ATG GAC AGT GCG TCA GAC TGT GCG GCA
Asn Ala Gly Ser Cys Phe Thr Arg Pro Val Ser Gln Val Leu 972 AAC GCT GGG TCC TGT TTC ACA CGA CCA GTC AGC CAA GTT CTG
Gln Asn Arg Cys Gln Arg Thr Cys Asn Thr Cys Asp Cys Arg 1014 CAG AAC AGG TGT CAA AGG ACA TGC AAT ACA TGC GAC TGC CGG
Asp Glu Ala Asn Asn Cys Ala Ala Ser Ile Asn Leu Cys Gln 1056 GAC GAG GCT AAC AAT TGT GCA GCT TCA ATT AAC CTC TGC CAG
Asn Pro Thr Phe Glu Pro Leu Val Arg Asp Arg Cys Gln Lys 1098 AAC CCC ACA TTT GAA CCT TTA GTT CGT GAT CGA TGC CAA AAG
Thr Cys Gly Leu Cys Ala Gly Cys Gly Phe Ile Ser Ser Gly 1140 ACT TGT GGT TTG TGT GCG GGG TGC GGA TTC ATC AGT AGC GGA
Figure 3.13a (cont.) Amino acid sequence of pET42/TES-26 construct
* Nucleic acid sequences in bold represent the GST-tag.
Ile Val Pro Leu Val Val Thr Ser Ala Pro Ser Arg Arg Val
148 1182 ATC GTT CCA TTA GTT GTG ACG AGT GCA CCT TCA AGA CGC GTG
Ser Val Thr Phe Ala Asn Asn Val Gln Val Asn Cys Gly Asn 1224 AGT GTG ACC TTC GCT AAC AAC GTT CAG GTG AAC TGC GGT AAT
Thr Leu Thr Thr Ala Gln Val Ala Asn Gln Pro Thr Val Thr 1266 ACT TTG ACG ACG GCG CAA GTA GCC AAC CAA CCG ACA GTA ACG
Trp Glu Ala Gln Pro Asn Asp Arg Tyr Thr Leu Ile Met Val 1308 TGG GAA GCG CAA CCG AAT GAC AGG TAC ACC CTC ATC ATG GTC
Asp Pro Asp Phe Pro Ser Ala Ala Asn Gly Gln Gln Gly Gln 1350 GAT CCT GAC TTC CCA AGC GCG GCG AAC GGC CAG CAG GGT CAG
Arg Leu His Trp Trp Val Ile Asn Ile Pro Gly Asn Asn Ile 1392 CGA CTT CAC TGG TGG GTA ATA AAC ATT CCA GGA AAT AAT ATC
Ala Gly Gly Thr Thr Leu Ala Ala Phe Gln Pro Ser Thr Pro 1434 GCC GGT GGT ACG ACG CTC GCA GCG TTC CAG CCA TCA ACA CCC
Ala Ala Asn Thr Gly Val His Arg Tyr Val Phe Leu Val Tyr 1476 GCC GCT AAT ACT GGT GTG CAC CGA TAC GTG TTT CTG GTA TAC
Arg Gln Pro Ala Ala Ile Asn Ser Pro Leu Leu Asn Asn Leu 1518 AGA CAG CCT GCC GCA ATA AAC TCT CCG CTA CTT AAT AAT TTG
Val Val Gln Asp Ser Glu Arg Pro Gly Phe Gly Thr Thr Ala 1559 GTA GTG CAG GAT TCG GAG CGG CCA GGT TTT GGT ACG ACT GCC
Phe Ala Thr Gln Phe Asn Leu Gly Ser Pro Tyr Ala Gly Asn 1601 TTT GCC ACT CAG TTT AAC CTT GGA TCA CCC TAT GCT GGC AAT
stop codon of gene
Phe Tyr Arg Ser Gln Ala *** 1643 TTC TAT CGA TCG CAG GCC TAA
Figure 3.13a (cont.) Amino acid sequence of pET42/TES-26 construct
* Nucleic acid sequences in bold represent the GST-tag.
149 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln 1 ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA
Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu 46 CCC ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys 91 CAT TTG TAT GAG CGC GAT GAA GGT GAT AAA TGG CGA AAC AAA
Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile 136 AAG TTT GAA TTG GGT TTG GAG TTT CCC AAT CTT CCT TAT TAT ATT
Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr 181 GAT GGT GAT GTT AAA TTA ACA CAG TCT ATG GCC ATC ATA CGT TAT
Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg 226 ATA GCT GAC AAG CAC AAC ATG TTG GGT GGT TGT CCA AAA GAG CGT
Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr 271 GCA GAG ATT TCA ATG CTT GAA GGA GCG GTT TTG GAT ATT AGA TAC
Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys 316 GGT GTT TCG AGA ATT GCA TAT AGT AAA GAC TTT GAA ACT CTC AAA
Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu 361 GTT GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG AAA ATG TTC GAA
Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr 406 GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT GAT CAT GTA ACC
His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr 451 CAT CCT GAC TTC ATG TTG TAT GAC GCT CTT GAT GTT GTT TTA TAC
Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe 496 ATG GAC CCA ATG TGC CTG GAT GCG TTC CCA AAA TTA GTT TGT TTT
Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys 541 AAA AAA CGT ATT GAA GCT ATC CCA CAA ATT GAT AAG TAC TTG AAA
Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr 586 TCC AGC AAG TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC ACG
Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 631 TTT GGT GGT GGC GAC CAT CCT CCA AAA TCG GAT GGT TCA ACT AGT
Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg 679 GGT TCT GGT CAT CAC CAT CAC CAT CAC TCC GCG GGT CTG GTG CCA CGC
Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe 724 GGT AGT ACT GCA ATT GGT ATG AAA GAA ACC GCT GCT GCT AAA TTC
Glu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly 769 GAA CGC CAG CAC ATG GAC AGC CCA GAT CTG GGT ACC GGT GGT GGC
Figure 3.13b Amino acid sequence of pET42/TES-32 construct
150 Ser Gly Ile Glu Gly Arg Gly Ser Met Ala Ile Ser Gly Ile Gln 814 TCC GGT ATT GAG GGA CGC GGG TCC ATG GCG ATA TCG GGG ATC CAG
start codon of the gene
Asn Ser Met Ile Ala Ala Ile Val Val Leu Leu Cys Leu His Leu 859 AAT TCC ATG ATC GCC GCA ATC GTC GTT TTG TTA TGT CTC CAC CTC
Ser Ala Thr Asn Ala Cys Ala Thr Asn Asn Asp Cys Gly Ile Phe 904 TCC GCC ACC AAT GCT TGC GCC ACC AAC AAT GAC TGT GGT ATT TTC
Gln Val Cys Val Asn Asn Val Cys Val Ala Asn Asn Gln Gly Cys 949 CAA GTG TGC GTT AAC AAC GTC TGC GTT GCC AAT AAC CAA GGA TGC
Asn Pro Pro Cys Val Pro Pro Gln Val Cys Val Ala Pro Met Cys 994 AAT CCG CCT TGT GTG CCT CCG CAG GTT TGC GTT GCA CCA ATG TGT
Val Ala Pro Pro Pro Ala Ala Thr Thr Thr Ala Ala Pro Gly Val 1039 GTC GCC CCT CCA CCG GCA GCC ACT ACA ACT GCA GCT CCA GGC GTT
Thr Thr Thr Arg Pro Arg Ala Cys Pro Pro Asn Trp Thr Pro Phe 1084 ACA ACT ACA AGA CCA CGT GCC TGC CCA CCC AAT TGG ACG CCG TTC
Asn Asn Asn Cys Tyr Ile Ala Ser Leu Pro Gly Arg Phe Leu Phe 1129 AAC AAC AAT TGC TAT ATC GCC TCT CTA CCT GGA CGC TTC CTT TTC
Asn Gln Ala Ser Asp Trp Cys Thr Gln Thr Gly Ser Arg Val Val 1174 AAC CAA GCT AGC GAC TGG TGT ACA CAG ACT GGC TCA AGA GTT GTG
Trp Phe Asp Gln Ser Thr Val Gly Asn Phe Gly Ser Glu Leu Asn 1219 TGG TTC GAC CAG TCG ACT GTA GGA AAC TTC GGC AGC GAA CTG AAC
Phe Val Asn Ser Phe Ala Leu Gly Arg Gly Val Thr Arg Tyr Trp 1264 TTC GTC AAC AGT TTC GCT CTC GGT CGT GGA GTA ACC AGG TAC TGG
Ile Gly Val Asn Arg Gln Phe Gly Gln Trp Val Phe Thr Asn Gly 1309 ATA GGA GTG AAC AGA CAG TTC GGC CAG TGG GTG TTT ACG AAT GGA
Ser Pro Val Ile Phe Ser Asn Trp Arg Pro Ser Gln Pro Asp Gly 1354 AGT CCA GTG ATA TTT TCG AAC TGG AGG CCT AGT CAA CCG GAC GGA
Cys Cys Gly Ser Asn Val Thr Cys Ala Phe Val Asn Tyr Ala Asn 1399 TGC TGT GGT TCC AAC GTC ACG TGT GCG TTC GTT AAT TAC GCG AAC
Phe Leu Gly Gln Trp Asp Asp Ala Pro Cys Gly Ser Leu Phe Thr 1489 TTC CTC GGC CAA TGG GAT GAT GCC CCA TGC GGA AGT TTG TTT ACG
Thr Pro Gln Gly Phe Val Cys Lys Arg Pro Leu *** 1505 ACT CCA CAA GGC TTC GTA TGC AAG AGA CCT CTC TAG
Figure 3.13b (cont.) Amino acid sequence of pET42/TES-32 construct
* Nucleic acid sequences in bold represent the GST-tag.
151 1 ATG TCG TAC TAC CAT CAC CAT CAC CAT CAC GAT TAC GAT ATC CCA Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro
46 ACG ACC GAA AAC CTG TAT TTT CAG GGC GCC ATG GAT CCG GAA TTC Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe
start codon of the gene
91 GCC CTT ATG CAC GTC CTT ACC GTC GCT CTC GTC GCA GTG CTA ATC Ala Leu Met His Val Leu Thr Val Ala Leu Val Ala Val Leu Ile
136 TGC GTT GCT ACA CCA CAA ATG ATG TCC AGT TCC TCT TCA TCG TCT Cys Val Ala Thr Pro Gln Met Met Ser Ser Ser Ser Ser Ser Ser
181 CCG TCA ACA TCC TCA TCG TCA GCG TCA ACA TCC TCA TCG TCC GCG Pro Ser Thr Ser Ser Ser Ser Ala Ser Thr Ser Ser Ser Ser Ala
226 TCA ACA TCC TCA TCG TCC GCG TCA ACA TCC TCA TCG TCC GCG TCA Ser Thr Ser Ser Ser Ser Ala Ser Thr Ser Ser Ser Ser Ala Ser
271 ACA TCC TCA TCG CCC GCG TCA ACA TCC TCA TCG TCC GCG TCA ACA Thr Ser Ser Ser Pro Ala Ser Thr Ser Ser Ser Ser Ala Ser Thr
316 AGC TCA ATG GCA GGA TCT ACA TCC ACA GCA GCT GGG CCG ACA TCC Ser Ser Met Ala Gly Ser Thr Ser Thr Ala Ala Gly Pro Thr Ser
361 AGC TCT TCT GTT TCC ACA TCA ACT CCA GCG GTG ATG ACA ACG ACG Ser Ser Ser Val Ser Thr Ser Thr Pro Ala Val Met Thr Thr Thr
406 CCA GCG TGC ATC GAC ACC GCT AAC GAC TGC CAG CTG TTC ACG CCA Pro Ala Cys Ile Asp Thr Ala Asn Asp Cys Gln Leu Phe Thr Pro
451 CTC TGC TTC GTT CAG CCA TAC AGC AGA GCA ATA CAG GGA AGA TGC Leu Cys Phe Val Gln Pro Tyr Ser Arg Ala Ile Gln Gly Arg Cys
496 CGA AGG ACG TGC AAC ATT TGC AGC TGT CAG GAC AGT GCC AAC GAC Arg Arg Thr Cys Asn Ile Cys Ser Cys Gln Asp Ser Ala Asn Asp
541 TGT GCA AAC TTT GTT TCA GTC TGC CTG AAC CCG ACC TAT CAG CCA Cys Ala Asn Phe Val Ser Val Cys Leu Asn Pro Thr Tyr Gln Pro
586 GTG CTT CGA TCA AGA TGC CCA CTG ACG TGC GGC TTC TGT AAG GGC Val Leu Arg Ser Arg Cys Pro Leu Thr Cys Gly Phe Cys Lys Gly
631 GAA TTC AAA GGC CTA CGT CGA CGA GCT CAA CTA GTG CGG CCG CTT Glu Phe Lys Gly Leu Arg Arg Arg Ala Gln Leu Val Arg Pro Leu
676 TCG AAT CTA GAG CCT GCA GTC TCG AGG CAT GCG GTA CCA AGC TTG Ser Asn Leu Glu Pro Ala Val Ser Arg His Ala Val Pro Ser Leu
Stop codon
721 GCT GTT TTG GCG GAT GAG AGA AGA TTT TCA GCC TGA Ala Val Leu Ala Asp Glu Arg Arg Phe Ser Ala ***
152 Figure 3.13c Amino acid sequences of pPROExTMHTa /TES-120 construct
* Nucleic acid sequences in bold represent the sequence of histidine tag.
3.2.4 Protein expression
3.2.4.1 Small-scale expression
A small-scale expression was performed for each recombinant clones to find the optimized parameter for recombinant protein expressions, to determine the solubility of the expressed recombinant proteins and to confirm the presence of fusion tags (GST and 6xHis) in the recombinant proteins by Western blot technique.
3.2.4.1.1 Optimizing the expression of the rTES-26, rTES-32 and rTES-120
recombinant proteins
3.2.4.1.1.1 Induction of the target proteins at different induction time-point
The small-scale expression was prepared according to section 2.2.14. The expression of the recombinant clones were studied at different post-induction time-point to determine the optimum time-point needed for optimum expression. Cells were induced with 1.0 mM IPTG at 30ºC for 0, 1, 2, 3, 4, 5, and 6 hours. Cells grown without IPTG were included as control.
The pET42 version “a” and “b” and pPROExTMHTa vectors were also included to see whether the vector itself could express protein. The loading volume for SDS-PAGE was standardized based on the total protein concentration (20 μg per lane). The protein molecular weight marker with a mixture of 7 purified proteins in the range of 14.4 to 116.0 kDa
(Fermentas, USA) was included.
The SDS-PAGE profiles for rTES-26, rTES-32 and rTES-120 recombinant proteins are shown in figure 3.14 a, b and c, respectively. A very small amount of protein was expressed in the control sets (lane 1) even though no IPTG was added (uninduced sample) for all sets of optimizations.
153 116.0 kDa 66.2 kDa ~ 70 kDa
45.0 kDa
35.0 ka
25.0 kDa
18.4 kDa
M 1 2 3 4 5 6 7 8
Figure 3.14a SDS-PAGE analysis of rTES-26 recombinant protein at different post- induction time-point
Lane M : Protein Molecular Weight Marker (Fermentas, USA) Lane 1 : Uninduced clone Lane 2 : 1 hour post-induction Lane 3 : 2 hours post-induction Lane 4 : 3 hours post-induction (optimum) Lane 5 : 4 hours post-induction Lane 6 : 5 hours post-induction Lane 7 : 6 hours post-induction Lane 8 : pET 42b vector (6 hours post-induction)
Note: Arrow shows the expressed recombinant protein. 10% SDS-PAGE was used.
154 116.0 kDa 66.2 kDa ~ 64 kDa 45.0 kDa
35.0 ka
25.0 kDa
18.4 kDa 14.4 kDa
M 1 2 3 4 5 6 7 8
Figure 3.14b SDS-PAGE analysis of rTES-32 recombinant protein at different post- induction time-point
Lane M : Protein Molecular Weight Marker (Fermentas, USA) Lane 1 : Uninduced clone Lane 2 : 1 hour post-induction Lane 3 : 2 hours post-induction Lane 4 : 3 hours post-induction Lane 5 : 4 hours post-induction (optimum) Lane 6 : 5 hours post-induction Lane 7 : 6 hours post-induction Lane 8 : pET 42a vector (6 hours post-induction)
Note : Arrow shows the expressed recombinant protein. 10% SDS-PAGE was used.
155 116.0 kDa 66.2 kDa 45.0 kDa
35.0 ka ~ 31 kDa 25.0 kDa
18.4 kDa
14.4 kDa
M 1 2 3 4 5 6 7 8
Figure 3.14c SDS-PAGE analysis of rTES-120 recombinant protein at different post- induction time-point
Lane M : Protein Molecular Weight Marker (Fermentas, USA) Lane 1 : Uninduced clone Lane 2 : 1 hour post-induction Lane 3 : 2 hours post-induction Lane 4 : 3 hours post-induction Lane 5 : 4 hours post-induction Lane 6 : 5 hours post-induction (optimum) Lane 7 : 6 hours post-induction Lane 8 : pPROExTMHTa vector (6 hours post-induction)
Note : Arrow shows the expressed recombinant protein. 10% SDS-PAGE was used.
156 The estimation of expected target proteins (shown with arrow) was based on the calculation below:
Number of base at ORF (bp) x 126.7 dalton
3
For expressions of rTES-26 and rTES-32, the yield of the recombinant proteins expressed (as determined by the thickness & intensity of the band) (~70 kDa and ~64 kDa, respectively) increases as the incubation time increases from 1 to 3 hours (for rTES-26; figure 3.14a) and from 1 to 4 hours (for rTES-32; figure 3.14b). For rTES-26, the yield of the expressed recombinant protein was approximately the same after 3 hours of incubation
(figure 3.14a), while for rTES-32, the yield of the expressed recombinant protein was approximately the same after 4 hours (figure 3.14b) The pET42 vector did not express any protein at the expected size of recombinant protein even at 6 hours post-induction (lane 8, figures 3.14a and b).
The yield of the expressed rTES-120 protein (~ 31 kDa) increases as the incubation time increases from 1 hour to 5 hours (figure 3.14c). The yield of rTES-120 recombinant protein expressed was approximately the same after 5 hours incubation (lanes 6 and 7).
Similar to the pET42 vector, pPROExHT vector also did not express any protein at the expected size of recombinant protein even at 6 hours post-induction (lane 8; figure 3.14c).
SDS-PAGE analysis revealed that 3, 4 and 5 hours post-induction were the optimum time- points for the expression of rTES-26, rTES-32 and rTES-120, respectively since the target bands produced at these hours were the most thickest and most prominent.
3.2.4.2 Determination of the solubility target protein and confirmation of presence of
protein tags
This study was performed to determine whether the recombinant protein is expressed in the cytoplasm or in the periplasm as inclusion bodies and to confirm the presence of GST and
157 histidine tags. Each recombinant protein was expressed following the optimized parameters as mentioned earlier. To the cell pellet, 24 parts of lysis buffer and 1 part of a cocktail of protease inhibitors were added, followed by the addition of lysozyme (final concentration of
0.5 mg/ml). The cell suspension was incubated on ice for 30 minutes, followed by lysing using French press 40K (Thermo Spectronic, USA) with a hydraulic pressure of 1700 psi
(the ratio selector set to the HIGH position). French Pressure Cell was placed on a 3-column filling stand. The samples were then poured (up to 35 ml/cell capacity) directly into the cell bore. The press uses an external hydraulic pump to drive a piston within a larger cylinder which contains the sample. The highly pressured sample was then squeezed passed a needle valve. The inclusion bodies were pelleted by centrifugation at 10,000 x g, 4ºC, for 30 minutes and the clear supernatant was carefully transferred into a sterile tube. SDS-PAGE analysis was performed (section 2.2.12) on the supernatant and pellet of lysed cells
(containing insoluble protein) and electroblotted onto a nitrocellulose membrane as described in section 2.2.13.1 to check the ‘site’ where the recombinant protein was found, whether it was present as soluble form in the supernatant or as insoluble inclusion bodies in the pellet.
3.2.4.2.1 Determination of histidine-tagged in rTES-120 recombinant protein by
Western blot analysis
The rTES-120 was resolved on a 10% SDS-PAGE gel (section 2.2.12) and electroblotted onto a nitrocellulose membrane as described in section 2.2.13.1. In order to check for the presence of the six-histidine fusion in the rTES-120 recombinant protein, the membrane strips were probed with mouse monoclonal anti-histidine-horseradish peroxidase (HRP) antibody (SantaCruz, USA) which was diluted at 1:300 for 2 hours. The strips were then washed thoroughly with TBST (10 minutes x 6) and developed using BM
Chemiluminescence substrate as described in section 2.2.13.2.4.
The rTES-120 recombinant protein displayed a distinctive and thick band at the expected molecular weight of approximately 31 kDa (shown with arrow) when reacted with
158 mouse monoclonal anti-histidine-HRP antibody (figure 3.15). This indicated that the expressed protein was tagged with polyhistidine. Western blot analysis showed that the amount of expressed rTES-120 target protein was present in greater amount in supernatant
(soluble protein) of the lysed cells (lane 2) as compared to that present in the pellet
(insoluble protein) (lane 1). The recombinant protein was then purified using metal affinity column technique. The procedure for recombinant protein purification will be discussed in chapter 4.
159 66.2 kDa 45.0 kDa 35.0 ka 25.0 kDa 31 kDa
M 1 2
Figure 3.15 Western blot analysis of rTES-120 recombinant protein expressed in supernatant and pellet
Lane M : Protein Molecular Weight Marker (Fermentas, USA) Lane 1 : Pellet of TES-120 lysate Lane 2 : Supernatant of TES-120 lysate
Note : Arrow shows the expressed histidine-tagged recombinant protein. 10% SDS-PAGE was used.
160 3.2.6.2.2 Determination of GST-tagged in rTES-26 and rTES-32 recombinant
proteins by Western blot analysis
The rTES-26 and rTES-32 recombinant proteins were resolved on 10% SDS-PAGE gel
(section 2.2.9.3) and electroblotted onto a nitrocellulose membrane as described in section
2.2.10.1. In order to check for the presence of the GST-tag in the recombinant proteins, the membrane strips were probed with mouse monoclonal anti-GST antibody (Novagen,
Germany) which was diluted at 1:10,000 for 1 hour. The strips were then washed thoroughly with TBSTT (5 minutes x 2) and TBS (5 minute x 1), followed by incubation with goat anti- mouse IgG-HRP (Santa Cruz, USA) at 1:5000 for 1 hour. The strips were again washed thoroughly with TBSTT (5 minutes x 5) and developed using BM Chemiluminescence substrate as described in section 2.2.10.2.
Figures 3.16 and 3.17 showed the pattern of rTES-26 and rTES-32 fusion proteins, respectively. The TES-26 and TES-32 lysates displayed a distinctive and thick band at the expected molecular weight of approximately 70 and 64 kDa, respectively when reacted with mouse monoclonal anti-GST antibody. This indicated that the expressed proteins were fused with GST. Western blot analysis also showed that the amount of expressed rTES-26 and rTES-32 target proteins were present in greater amount in supernatant (soluble protein) of the lysed cells (lane 2) as compared to that present in the pellet (insoluble protein) (lane 1).
The recombinant proteins were then purified using affinity column technique using GST
Bind Resin. The procedure for recombinant protein purification will be discussed in chapter
4.
161 100 kDa 75 kDa ~70 kDa 50 kDa 40 kDa
30 kDa
25 kDa 20 kDa
M 1 2
Figure 3.16 Western blot analysis of rTES-26 recombinant protein expressed in supernatant and pellet
Lane M : Protein Molecular Weight Marker (Fermentas, USA) Lane 1 : Pellet of TES-26 lysate Lane 2 : Supernatant of TES-26 lysate
Note : Arrow shows the expressed GST-tagged recombinant protein. 10% SDS-PAGE was used.
162 100 kDa 75 kDa ~ 64 kDa 50 kDa 40 kDa 30 kDa 25 kDa 20 kDa
10 KDa
M 1 2
Figure 3.17 Western blot analysis of TES-32 recombinant protein expressed in supernatant and pellet
Lane M: Protein Molecular Weight Marker (Fermentas, USA) Lane 1: Pellet of TES-32 lysate Lane 2: Supernatant of TES-32 lysate
Note : Arrow shows the expressed GST-tagged recombinant protein. 10% SDS-PAGE was used.
163 3.3 Discussion
In the present study, three genes which encode TES-26, TES-32 and TES-120 proteins were selected for cloning and expression. The ORF of the gene which encodes for TES-120 protein was chosen because it is abundantly expressed by T. canis larvae and is highly antigenic (Gems & Maizels, 1996; Tetteh et al., 1999). As mentioned before, the previously produced rTES-120 protein was insoluble and IgG assay was employed in the Western blot analysis (Fong et al., 2003; 2004). However, in this study different expression system and immunoassay detection (IgG4 assay) was employed, the former enabled production of soluble protein and the latter led to the development of a more specific assay. To date, the antigen produced by gene encoding TES-26 has not been reported to have a diagnostic potential for serodiagnosis. However, appearance of a 26 and 32 kDa protein bands are being used as serodiagnostic markers for confirmation of human toxocariasis in a commercial
TES-based Western blot IgG kit (Testline, USA), thus we hypothesized that rTES-26 and rTES-32 would be useful diagnostic reagents.
This study described the cloning via RT-PCR, sequencing and expression of the
ORF of the genes encoding TES-26, TES-32 and TES-120. RT-PCR method was used to amplify cDNA copies of mRNA and ‘gene’-specific downstream primers were used to prime cDNA synthesis. Complementary DNA synthesis and PCR took place successively in a specially optimized RT-PCR buffer during an uninterrupted thermal-cycling program. Since the reaction tube was not handled between cDNA synthesis and PCR amplification, the
StrataScript One-Tube RT-PCR system involves less hands-on time, thus this reduced the risk of sample contamination when compared to a two tubes system. A negative control reaction in which StrataScript was omitted was included to ensure that the RT-PCR product was the result of amplification of the cDNA template and not the genomic DNA. This system can readily detect RNA target of 0.1 – 10 ng of poly (A)+ RNA.
The high quality, purity and integrity of intact RNA is very crucial for successful synthesis of cDNA. The widest use of poly(A)+ RNA is for cDNA synthesis, either for RT-
PCR or cloning purposes. For most applications, total RNA is sufficient, but in some cases
164 sensitivity of mRNA (poly(A)+ RNA) is beneficial especially in construction of cDNA library. The most important is to maintain an RNase-free environment starting with RNA isolation and continuing through analysis to ensure success because RNAse and DNAse can cause degradation of RNA.
In this study, the concentration of mRNA at the first preparation was too diluted and could not be measured by biophotometer. However, in the second preparation the concentration was 1.6 µg/µl. Even when the concentration of mRNA was too diluted, it could still be amplified by StrataScript One-Tube RT-PCR kit. The purity of the mRNA was
2.0 (ratio of A260: A280 values should fall in the range of 1.8 – 2.1) for both of the preparations. High-quality intact RNA is essential for successful synthesis of cDNA.
In this study, the amplification of T. canis genes were performed using the Easy-ATM high-fidelity polymerase which is 6-fold more accurate than Taq DNA polymerase for high reliability amplification. In addition to the exonuclease activity that improves PCR fidelity, the Easy-A enzyme (in the Easy-ATM polymerase) possesses terminal transferase activity, which adds 3’-A overhangs, allowing the amplicons generated to be easily cloned into any
TA cloning vector. Although, the most commonly used enzyme is Taq DNA polymerase that adds adenine to the 3’ ends of PCR products, it lacks proofreading (3’ to 5’ exonuclease activity) for accurate amplification. Taq DNA polymerase possessed the highest error rate with the highest percentage of mutated PCR products after amplification of a target sequence. Error rate of approximately 1 per 250 nucleotides has been reported (Kunkel,
2004). The use of proofreading or high fidelity DNA polymerase in PCR cloning is essential in order to minimize the introduction of errors generated during the PCR synthesis.
The construction of TES-26, TES-32 and TES-120 gene fragments had been successfully performed and cloned into pCR2.1 TOPO vector. The linearized vector has a single overhang 3’-thymidine residue, which allows PCR inserts to ligate efficiently with the vector through A-T complementary pairing. However, sequence analysis revealed that the
TOPO/TES-26 gene fragment had four mutations with respect to the reported sequence. The nucleotide changes resulted in changes of amino acids of the protein. The nucleotide changes
165 might be due to replication errors during PCR synthesis. In-vitro site directed mutagenesis was performed to correct the base-errors in TOPO/TES-26 clone. PCR-based site-directed mutagenesis system used increased template concentration and reduced cycling number to decrease potential second-site mutations during the PCR. The Dpn1 endonuclease (target sequence: 5’-GM6ATC-3’) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and to select for mutation-containing amplified DNA (Stratagene handbook, 2003). DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpn1 digestion. Pfu DNA polymerase was used prior to end-to-end ligation of the linear template to remove any bases extended onto the 3’ ends of the product by PfuTurbo® DNA polymerase. The recircularized and nicked vector DNA incorporating the desired mutations was then ligated and transformed to E. coli. In this study, three mutated bases of TOPO/TES-26 recombinant DNA were successfully repaired at the first attempt. However, several attempts were required before the fourth mutated base was finally repaired.
The TOPO/TES-32 and TOPO/TES-120 DNA sequences analysis revealed that no amplification error (corresponding 100% identity to their reported sequences in the
Genbank) was generated during amplification process. The DNA sequence results confirmed that the ORF of the genes encoding TES-26 and TES-32 were in the correct orientation however, ORF of the gene encoding TES-120 was in the antisense orientation since the sense strand of the cloned fragment lies on the opposite strand from the vector’s promoter.
However, this condition did not affect subcloning into an expression vector since digestion with EcoR1 enzyme would generate a fragment with identical sticky ends, enabling a good chance of obtaining recombinant plasmids in the sense orientation in the pPROExTMHT bacterial expression vector.
The DNA sequences were compared with pET42 and pPROExTMHT expression vectors to check for restriction enzyme sites and version suitability that can be used for protein expression. TES-26, TES-32 and TES-120 fragments with sticky ends were
166 generated by EcoR1 digestion and ligated into pET42b, pET42a and pPROExTMHTa vectors respectively, which had been predigested using the same restriction enzyme.
The pET42 and pPROExTMHT vectors were chosen as expression vectors in this study since they allow efficient purification and detection of expressed fusion proteins. The affinity tags (His and GST) in these vectors will facilitate purification, improve yield and increase solubility. The pET expression system was used in 90% of the protein preparation systems in the Protein Data Bank (PDB) for the year 2003. The pET-42 series are tagged with the 220 amino acid GST•TagTM protein as a fusion partner. When expressed in the soluble form, GST•Tag fusion proteins can be purified with immobilized glutathione. The pPROExHT prokaryotic expression vectors are tagged with the 6 histidine sequence (His)6, located at the amino terminus of the fusion protein which facilitates binding of the expressed protein to nickel resin during protein purification. The presence of trc promoter and lacIq gene (in pPROExHT vectors) and lacI (in pET42 vectors) enable inducible expression of a cloned gene using IPTG as inducer.
The pET42 version b and a, and pPROExTMHT version a vectors were chosen because the sequences showed that DNA inserts could be positioned in frame with the initiation ATG, fusion partners and stop codon. The presence and orientation of the DNA sequences of TES-26 and TES-32 in pET and TES-120 in pPROExTMHT constructs were checked by performing PCR with the vector and ‘gene’-specific primers and confirmed by
DNA sequencing. The size of the inserts was further verified by restriction enzymes digestions. The pET42/TES-26, pET42/TES-32 and pPROExTMHT/TES-120 DNA sequences analysis further confirmed that recombinant DNA sequences of TES-26, TES-32 and TES-120 were in the correct reading frames, with the presence of appropriate fusion tags and stop codons.
The recombinant DNA was maintained in E. coli TOP10 host that is deficient in T7
RNA polymerase. After the plasmids were established in a non-expression host, they were transformed into a host bearing the T7 RNA polymerase gene (DE3 lysogen). BL21 is deficient in the lon protease and lacks the ompT outer membrane protease that can degrade
167 proteins during purification (Studier et al., 1990). Thus, target proteins should be more stable in BL21 than in host strains that contain these proteases.
Once the cloning has been successful, a good starting point for any expression is to determine the solubility of the target protein. Small-scale expression was performed before proceeding with a large scale production. The use of small expression cultures provides a rapid way to study the different growth conditions on expression levels and solubility of target proteins.
After the recombinant plasmids (TES-26, TES-32 and TES-120) were established in a λDE3 lysogen, expression of the recombinant DNA was induced by the addition of IPTG to a growing culture. T7 RNA polymerase is very selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression. The desired product can comprise more than 50% of the total cell protein within a few hours after induction. For pET and pPROExTMHT constructions carrying the T7lac promoter, 1 mM
IPTG is recommended and was used for the proteins expression of TES-26, TES-32 and
TES-120. After IPTG induction, the temperature was reduced to 30ºC in order to decrease the rate of protein synthesis, which promote higher yield of soluble proteins. Even in the absence of IPTG (uninduced state), some expression of T7 RNA polymerase from the lacUV5 promoter in λDE3 lysogens was observed due to the leaky expression of T7 RNA polymerase.
Pilot study on the protein expressions revealed that 3, 4 and 5 hours of post- induction at 30°C with 1 mM IPTG was optimal for expression of rTES-26, rTES-32 and rTES-120 recombinant proteins, respectively. The optimized conditions were then used for large-scale production and purification of recombinant proteins (discussed in chapter 4). The immunoreactivity study by Western blot analysis on the TES-26, TES-32 and TES-120 lysates (figures 3.15, 3.16 and 3.17) showed that these recombinant DNA were fused to GST
(TES-26 and TES-32) and histidine tags (TES-120). Thus, in this study, ORF of the genes encoding TES-26, TES-32 and TES-120 were successfully cloned into pET-42 and pPROExTMHT vectors respectively.
168 CHAPTER FOUR
PURIFICATION OF rTES-26, rTES-32 AND rTES-120 RECOMBINANT
PROTEINS IN E. coli
4.1 Introduction
In the previous study, the ORF of the genes encoding TES-26, TES-32 and TES-120 were successfully cloned and expressed in the appropriate bacterial expression vectors for production of the respective recombinant proteins. This chapter describes the large-scale expression and affinity purification of these three recombinant proteins.
Affinity purification is based on the specific interaction of a target molecule with an immobilized ligand. The addition of fusion tags such as polyhistidine and GST to the target proteins using appropriate expression vectors enables affinity purification by a number of strategies (Baneyx, 1999; Terpe, 2003). The proteins are able to purify under gentle, native
(non-denaturing) conditions, or in the presence of 6M guanidine or 8M urea (denaturing conditions). The decision whether to purify the recombinant protein under native or denaturing conditions depends on the solubility of the protein. Since in chapter 3, the rTES-
26, rTES-32 and rTES-120 recombinant proteins were shown to be abundantly expressed in soluble form in the cytoplasm of the E. coli, these proteins were subsequently purified under native conditions.
The goal of the experiments in this chapter is to produce rTES-26, rTES-32 and rTES-120 recombinant proteins in high concentration and high purity to be used for the development of ELISA for the detection of human toxocariasis. The approaches applied in the recombinant protein purifications are shown in the figure 4.1.
169 Expression of T. canis recombinant proteins in large culture volumes
Purification of rTES-120 histidine- Purification of rTES-26 and rTES-32 tagged recombinant protein under GST-tagged recombinant proteins native conditions under native conditions
Laboratory-scale purification of rTES-26 and rTES-32 recombinant proteins using batch/gravity flow
Optimization of Fast Protein Liquid Chromatography (FPLC) method using linear gradient 0 – 100% • Optimization of salt concentrations (0.3 M, 0.5 M)) • Optimization of washing column Optimization of proteolytic cleavage of volume (5CV, 10CV) GST-Tag using Factor Xa • Optimization of Factor Xa concentrations (0.1, 0.2, 0.5 U/ul) • Optimization of incubation times (2, 4, 8, 16 hours) FPLC protein purification using optimized parameters
Up-scaling of proteolytic cleavage of GST- Tag using optimized parameters
Removal of Factor Xa
Immunoreactivity and specificity study of the purified recombinant proteins by Western blot analysis
Figure 4.1 Overview strategies for purification of the T. canis recombinant proteins under native conditions
170 4.2 Methodology
4.2.1 Purification of rTES-120 histidine-tagged recombinant protein
4.2.1.1 Large scale protein expression
The rTES-120 recombinant protein was expressed in a large volume culture using the optimized parameters (section 3.2.4.1.1). For this purpose, two liters (four 500 ml culture each in 1L flask) of Terrific broth culture of the recombinant bacteria was used to express the recombinant proteins. The culture was incubated at 37ºC, 200 rpm until the OD600 reached the mid-log phase of growth i.e. OD600 = 0.5. The recombinant bacteria were then induced with IPTG to a final concentration of 1 mM. The incubation was continued at a reduced temperature of 30ºC, 200 rpm for 5 hours, then the culture was harvested by centrifugation at 10000 x g, 4ºC for 15 minutes. The supernatant was decanted and cell pellet was stored at -80ºC.
4.2.1.2 Preparation of cleared cell lysates under native conditions
The cell pellet (from section 4.2.1.1) was removed from -80ºC, thawed on ice for 15 minutes and weighed. The cell pellet was resuspended in lysis buffer at 1.5 ml per gram of wet cell weight. Cells were lysed under native conditions (described below) and were kept on ice throughout the process. To the cell pellet, 24 parts of lysis buffer and 1 part of a cocktail of protease inhibitors were added, followed by the addition of lysozyme (final concentration of
0.5 mg/ml). The cell suspension was mixed by pipetting up and down and incubated on ice for 30 minutes, followed by lysing using French press 40K (Thermo Spectronic, USA) with a hydraulic pressure of 1700 psi (the ratio selector set to the HIGH position). French
Pressure Cell was placed on a 3-column filling stand. The samples were then poured (up to
35 ml/cell capacity) directly into the cell bore. The press uses an external hydraulic pump to drive a piston within a larger cylinder which contains the sample. The highly pressured solution was then squeezed passed a needle valve.
The cell suspension was then centrifuged (Hitachi CP 80 MX, USA) at 10,000 x g,
4ºC for 30 minutes to pellet the cellular debris. The supernatant was carefully transferred
171 into a sterile tube. DNase 1 (to a final concentration of 5 µg/ml) was then added, followed by incubation on ice for 10-15 minutes. The supernatant was centrifuged as above, the supernatant (cleared lysate) was filtered using 0.45 µm syringe filter prior to purification to prevent blocking of the column during purification process. The filtered supernatant was then immediately subjected to purification under native condition.
4.2.1.3 Optimization of rTES-120 recombinant protein purification using FPLC under
native condition
The affinity purification of rTES-120 histidine-tagged protein was performed under native conditions as described by the manufacturer (Amersham BioSciences, Sweden) since the rTES-120 recombinant protein was shown in chapter 3 to be expressed mainly in supernatant of the lysed cells (soluble form).
Two purification parameters were optimized, namely the concentration of salt in the lysis and washing buffers (0.3 and 0.5 M) and washing column volume (CV) [5 and 10
CVs]. FPLC protein purification was performed at room temperature using AKTATMprime
(Amersham BioSciences, Sweden) (figure 4.2). The AKTATMprime equipment was set up according to the protocol described in section 2.2.15.1. HisTrap HP 1 ml (Amersham
BioSciences, Sweden) prepacked, ready-to-use column containing precharged Ni
SepharoseTM High Performance was chosen as the matrix. The binding capacity of the resin is protein-dependent and lies between 5-10 mg/ml. The 1/16” 50 ml superloop was assembled according to the protocol described by the manufacturer.
The HisTrap HP column was washed using a continuous gradient of increasing imidazole concentration starting from 20 mM, 30 mM and finally 40 mM. Each washing step was performed at 5 or 10 column volumes. The washing buffer removed proteins that bind non- specifically to the resin. Finally, the purified recombinant protein was eluted with 10 column volumes of elution buffer containing 250 mM imidazole. The protein fractions were collected (500 µl/tube) once the chromatogram peak during elution started to increase and
172 C A B
G
E F D
Figure 4.2 FPLC protein purification was performed using AKTATMprime (Amersham BioSciences, Sweden)
A: 50 ml superloop; B: HisTrap HP 1 ml prepacked column; C: tube collector; D: sample injection; E: wash buffer; F: display menu, G: waste
173 the collection was terminated when the peak went down to baseline. The fractions were then analyzed by 10% SDS-PAGE (section 2.2.12).
4.2.2 Purification of rTES-26 and rTES-32 GST-tagged recombinant proteins
4.2.2.1 Large scale protein expression
The rTES-26 and rTES-32 recombinant proteins were expressed in a large volume using the optimized parameters (section 3.2.4.1.1). For this purpose, two liters (four 500 ml culture each in 1L flask) of Terrific broth culture for each of the two recombinant bacteria was employed. The cultures were incubated at 37ºC until the OD600 reached the mid-log phase of growth (OD600 = 0.5). The recombinant bacteria were then induced with IPTG to a final concentration of 1 mM. The incubation was continued at a reduced temperature of 30ºC for
3 hours for rTES-26 recombinant bacteria and 4 hours for rTES-32 recombinant bacteria.
The cultures were then harvested by centrifugation at 10 000 x g, 4ºC for 15 minutes. The supernatants from each of the culture were decanted and cell pellets were stored at -80ºC for further analysis.
4.2.2.2 Cell extracts preparation for native purification
This procedure was performed to isolate protein in the soluble fraction using mechanical disruption method as described by the manufacturer (Novagen, Germany).
The cell pellet (from 0.5 L culture) was removed from -80ºC and thawed on ice for
15 minutes. The cell pellet was resuspended in ice-cold 1X GST Bind/Wash buffer at 4 ml per 100 ml culture and the cells were subsequently lysed under native condition. To the cell pellet, 24 parts of lysis buffer and 1 part of protease inhibitors cocktail was added, followed by the addition of lysozyme (final concentration of 0.5 mg/ml). The cell lysate was incubated at 30ºC in a waterbath for 15 minutes, followed by lysis using French press 40K (Thermo
Spectronic, USA) as described in section 4.2.1.2. The cell lysate was then centrifuged using an ultracentrifuge (Hitachi CP 80 MX, USA) at 39,000 x g, 4ºC for 20 minutes to separate the cellular debris. DNase 1 (to a final concentration of 5 µg/ml) was then added, followed
174 by incubation on ice for 10-15 minutes. The supernatant was centrifuged as above and the supernatant (cleared lysate) was filtered using 0.45 µm syringe filter to prevent blocking of the column during purification process, then immediately used for purification under native condition.
4.2.2.3 Laboratory scale purification of GST-tagged proteins under native
conditions
The affinity purifications of these GST-tagged fusion proteins were performed under native condition as described by the manufacturer (Novagen, Germany) since the rTES-26 and rTES-32 recombinant proteins were present in sufficient amounts in the soluble form.
The purification process was performed at room temperature. The GST Bind Resin
(Novagen, USA) and buffer components were allowed to equilibrate at room temperature before use. The bottle of GST Bind Resin (supplied as 80% slurry in 20% ethanol) was gently mixed by inversion until completely suspended. The manual purification was set-up as shown in figure 4.3. The desired amount of slurry (1 ml slurry made up 0.5 ml settled bed/column volume) was transferred into a manual column (SizeSepTM 400 Spun Column,
Pharmacia BioTech, Sweden). The resin was allowed to pack under gravity flow. When the level of storage buffer (20% ethanol) drops to the top of the column bed, the resin was washed with 5 column volumes of GST Bind/Wash Buffer. The extract was rapidly brought to room temperature using a room temperature waterbath immediately before incubating with the resin for 30 minutes. The lysate-resin mixture was then carefully loaded into a column. The flow-through was collected. The column was washed with 10 column volumes of GST Bind/Wash Buffer. The wash buffer removed proteins that bind non-specifically to the resin. Finally, the purified recombinant protein was eluted with 5 volumes of elution buffer. Protein fractions of 500 µl (F1-F10) per fraction were collected and analyzed by 10%
SDS-PAGE (section 2.2.12).
175 15 ml tube attached to the Spun column
Retort stand Parafilm
Spun column
Collection tubes
Figure 4.3 Purification of rTES-26 and rTES-32 GST-tagged proteins using gravity flow
The Spun column was connected to the 15 ml tube and attached to retort stand. The resin was transferred into a Spun column and allowed to pack under gravity flow. The collection tubes were placed at the bottom of the column during protein elution.
176 4.2.2.4 Proteolytic cleavage of target protein from GST Tag
Restriction Grade Factor Xa (Novagen, Germany) is a highly purified enzyme isolated from bovine plasma and shows no secondary cleavage from contaminating proteases. It is a site- specific endoprotease that exhibits very low non-specific cleavage under many conditions and preferentially cleaves at the C-terminal peptide bond of its recognition sequence (Ile-
Glu-Gly-Arg) and can, therefore, be used for removing all vector-encoded sequences. The cleavage was performed as described by the manufacturer by using Factor Xa Cleavage
Capture kit (Novagen, Germany).
4.2.2.4.1 Small-scale optimization of proteolytic cleavage
Optimization of the cleavage conditions were performed in the small-scale reactions using the protocol as described by the manufacturer (Novagen, Germany). The small-scale optimization was performed to estimate the appropriate ratio of enzyme : target protein
(1:100, 1:50, 1:20) at different incubation times (2, 4, 8, 16 hours). The serial dilutions of
Factor Xa (0.1, 0.2, 0.5 U enzyme/μl) were made in Xa Dilution/Storage Buffer. The following reactions were assembled in four separate microfuge tubes: 5 μl of 10X Factor Xa
Cleavage/Capture Buffer, 10 μg of target protein, 1 μl of diluted Factor Xa (each tube received 1 μl of a different enzyme dilution; the last tube received 1 μl of Dilution/Storage
Buffer only as a negative control) and deionized water to a total volume of 50 μl. Parallel reaction using Xa Cleavage Control Protein (positive control) in the same buffer system was included to monitor the cleavage reaction. The mixture was incubated at room temperature
(20-21ºC) for 2, 4, 8 and 16 hours. At the end of each incubation times, 10 μl aliquots of each sample was removed and immediately mixed with SDS-PAGE sample buffer (2X) to completely quench Factor Xa Protease activity. The extent of cleavage in each sample was determined by SDS-PAGE analysis as described in section 2.2.12. The non-cleaved recombinant protein was used as a control to check the success of cleavage.
177 4.2.2.4.2 Scale-up of cleavage reaction
Once the optimal cleavage conditions have been found, the reaction was scaled up proportionally. Following the above protocol, 1 mg recombinant protein was digested in a total volume of 5 ml. The reaction was assembled as follows: 500 μl of 10X Factor Xa
Cleavage/Capture Buffer, 1 mg of target protein, 100 μl of diluted Factor Xa and deionized water to a total volume of 5 ml. The mixture was incubated at room temperature (20-21ºC) using the optimised time (as determined in 4.2.2.4.1).
4.2.2.4.3 Factor Xa capture after cleavage
After protease digestion of GST-tagged fusion proteins, Factor Xa was removed by affinity chromatography using Xarrest Agarose as described by the manufacturer (Novagen,
Germany). The Xarrest Agarose binds the protease in the reaction mixture while cleaved recombinant protein remains in solution. After centrifugation, the cleaved recombinant protein is recovered in the filtrate tube.
The sufficient amount of Xarrest Agarose to capture Factor Xa present in the cleavage reaction was determined based on the amount of Factor Xa to be captured (the binding capacity is 4 units Factor Xa per 50 μl settled bed volume (100 μl slurry) in 1X
Factor Xa Cleavage/Capture Buffer).
The Xarrest Agarose (supplied as 50% slurry) was mixed by gentle tumbling/agitation of the bottle until it was fully resuspended, then two settled resin volume was transferred to a centrifuge tube. The mixture was centrifuged at 1 000 x g for 5 minutes.
The supernatant was carefully discarded. Equilibration of the resin with 1X Factor Xa
Cleavage/Capture Buffer is necessary for maximum capture efficiency and prevents contamination of the cleaved recombinant protein with resin storage buffer. The agarose was resuspended in 10 settled resin volume of 1X Factor Xa Cleavage/Capture Buffer and was centrifuged as above. The supernatant was carefully discarded. Subsequently, one settled
178 resin volume of 1X Factor Xa Cleavage/Capture Buffer was added and fully resuspended by gently mixing.
The equilibrated Xarrest Agarose was transferred to a sample cup of 2-ml spin filter. The entire volume of cleavage reaction was added, gently mixed and incubated at room temperature for 5 minutes. The reaction was centrifuged as above to remove the Xarrest
Agarose. Bound Factor Xa was retained in sample cup, and cleaved target protein flowed into the filtrate tube during centrifugation. The cleaved protein was then stored at -80ºC for further use.
4.2.3 Determination of immunoreactivity of the purified TES-120 recombinant
proteins by Western blot technique
The antigenicity and specificity of the purified rTES-120 recombinant protein was determined by Western blot analysis as described in section 2.2.13. The purified recombinant protein (approximately 100 µg) was loaded in a 10% SDS-PAGE gel, with a protein marker of 14.4 to 116.0 kDa as the molecular weight marker (Fermentas, USA). The recombinant protein on the gel was then electroblotted onto nitrocellulose membrane as described in section 2.2.13.1. The membrane was cut into strips and incubated with mouse monoclonal anti-histidine-HRP antibody (as described in section 2.2.13.2.1) as well as sera from patients infected with toxocariasis, STH and healthy individuals. The latter strips were then probed with mouse anti-human IgG4 conjugated with HRP (described in section
2.2.13.2.3). The strips were then washed thoroughly (6 x 15 minutes) and developed using
BM Chemiluminescence substrate (described in section 2.2.13.2.4).
4.2.4 Determination of immunoreactivity of the cleaved rTES-26 and rTES-32
recombinant proteins by Western blot technique
The antigenicity and specificity of the cleaved rTES-26 and rTES-32 recombinant proteins were determined by Western blot analysis as described in section 2.2.13. Each of the cleaved recombinant protein (approximately 100 µg) was loaded in separate 10% SDS-PAGE gel,
179 with a protein marker of 14.4 to 97.0 kDa as the molecular weight marker (Amersham
BioSciences, Sweden). The cleaved proteins on the gel were then electroblotted onto nitrocellulose membranes as described in section 2.2.13.1. The membrane strips were then incubated with mouse monoclonal anti-GST antibody (described in section 2.2.13.2.2) as well as sera from patients infected with toxocariasis, STH and healthy individuals. The former strips were then probed with goat anti-mouse IgG conjugated with HRP and the latter strips were probed with mouse anti-human IgG4 conjugated with HRP (as described in section 2.2.13.2.3). The strips were then washed thoroughly (6 x 15 minutes) and developed using BM Chemiluminescence substrate as described in section 2.2.13.2.4.
4.3 Results
4.3.1 Optimization of TES-120 recombinant protein purification using FPLC under
native conditions
The concentration of each protein fraction (fraction 1 to fraction 20) obtained in each run is shown in Table 4.1. The output chromatogram result is shown in figure 4.4. Most of the
TES-120 histidine-tagged protein was eluted at fractions 10 to 20 (shown as increased peak during elution). Total protein eluted by this procedure was approximately 1.5 mg/L (in buffers containing 0.3 M NaCl for both 5 and 10 CV) and 1.25 mg/L (in buffers containing
0.5 M NaCl for both 5 and 10 CV).
SDS-PAGE gels of the protein fractions were visualized with Coomassie Blue staining as shown in figure 4.5. In figures 4.5a and b, the eluted target protein of approximately 31 kDa is shown in lane 1. The amount of eluted protein increased until a maximum amount was achieved in fraction 12 (lane 3). In figures 4.5c and d, the eluted target protein of approximately 31 kDa is shown in lane 3. The amount of eluted protein increased until a maximum amount was achieved in fractions 14 and 15 (lanes 5 and 6).
Purification using buffers containing 0.5 M NaCl at 5 CV was chosen as the optimum conditions. This was because there was no difference observed in protein yields between 5
180 and 10 CV and the fractions were less contaminated with other bacterial proteins when 0.5
M was employed as compared to 0.3 M NaCl.
Table 4.1 Concentrations of purified rTES-120 recombinant protein obtained by FPLC using different concentrations of salt and washing column volume
Elution Protein Protein Protein Protein Fractions concentration concentration concentration concentration (mg/ml) (mg/ml) (mg/ml) (mg/ml) 0.3 M/5CV 0.3 M/10CV 0.5 M/5CV 0.5 M/10CV F1 0.0 0.0 0.0 0.0 F2 0.0 0.0 0.0 0.0 F3 0.0 0.0 0.0 0.0 F4 0.0 0.0 0.0 0.0 F5 0.0 0.0 0.1 0.1 F6 0.0 0.0 0.25 0.1 F7 0.1 0.1 0.4 0.2 F8 0.1 0.1 0.6 0.45 F9 0.1 0.1 0.7 0.6 F10 0.2 0.19 1.0 0.8 F11 0.9 0.9 1.3 1.5 F12 1.2 1.1 0.9 1.3 F13 0.9 1.0 0.9 1.1 F14 0.9 0.9 1.2 1.1 F15 0.6 0.5 1.2 1.0 F16 0.3 0.3 0.7 0.9 F17 0.1 0.1 0.5 0.4 F18 0.1 0.1 0.1 0.1 F19 0.1 0.1 0.1 0.1 F20 0.1 0.1 0.1 0.1
Pooled 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/ml fractions (F11-F16) (F11-F16) (F13-F17) (F13-F17)
Total protein yield 1.5 mg/L 1.5 mg/L 1.25 mg/L 1.25 mg/L (in 1 litre of culture broth)
0.3 M, 0.5 M : Purification using buffer containing 0.3 and 0.5 M NaCl 5CV, 10 CV: Washing with 5 and 10 column volumes
Total protein yield (in 1 litre of culture broth): protein concentration of pooled fractions x total volume of pooled fractions litre of culture
For 0.3 M/5CV & 0.3 M/10CV; 1 mg/ml x 3 ml = 1.5 mg/L 2 L
181 For 0.5 M/5CV & 0.5 M/10CV; 1 mg/ml x 2.5 ml = 1.25 mg/L 2 L
182 Sample injection 2007Feb07no001:10_UV 2007Feb07no001:10_Fractions 2007Feb07no001:10_Inject 2007Feb07no001:10_Logbook
mAu
3000
2500
2000
1500 Wash buffer (40 mM)
1000 Wash buffer (30 mM) Sample elution
500 Wash buffer (20 mM)
Break point 2 Break point 3 Break point 4 Break point 5 Break point 6 Break point 8
0 13579 12 16 20 24 28 32 36 40 44 48 52 Waste 0 20 40 60 80 100 120 140 ml
Figure 4.4 The output chromatogram result of purification of rTES-120 recombinant protein using FPLC method
183 (a) Buffers containing 0.3 M NaCl (5 column volumes) (b) Buffers containing 0.3 M NaCl (10 column volumes)
116.0kDa 116.0kDa 66.2 kDa 66.2 kDa 45.0 kDa 45.0 kDa 35.0 kDa 35.0 kDa
30.0 kDa 30.0 kDa
18.4 kDa 18.4 kDa 14.4 kDa 14.4 kDa
M 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10
(c) Buffers containing 0.5 M NaCl (5 column volumes) (d) Buffers containing 0.5 M NaCl (10 column volumes)
116.0kDa 116.0kDa 66.2 kDa 66.2 kDa 45.0 kDa 45.0 kDa 35.0 kDa 35.0 kDa
30.0 kDa 30.0 kDa
18.4 kDa 18.4 kDa 14.4 kDa 14.4 kDa
M 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10
Figure 4.5 SDS-PAGE analysis of rTES-120 protein fractions collected using FPLC method
Arrow shows the expressed recombinant proteins. Lane M : Protein marker (Fermentas, USA); Lane 1- 10 : Protein fractions 10 to 19
184 4.3.2 Purification of rTES-26 and rTES-32 GST fusion proteins under native conditions
Figure 4.6(a) shows the SDS-PAGE profile of rTES-26 protein fractions. The rTES-26 recombinant protein of approximately 70 kDa was eluted in lanes 1 to 9. The amount of eluted protein increased until a maximum quantity was achieved (lane 2). Subsequently, the amount of eluted protein decreased until no more protein was eluted. The concentration of rTES-26 recombinant protein determined for fraction 1 to fraction 10 is shown in table 4.2.
The highest protein concentration was observed in fraction 2 with 1.0 mg/ml. Total protein eluted by this procedure was 4.0 mg/liter of culture.
Figure 4.6(b) shows that the rTES-32 recombinant protein of approximately 64 kDa which was eluted in lanes 1 to 9. The amount of eluted protein increased until a maximum quantity was achieved (lane 2). Subsequently, the amount of eluted protein decreased until no more protein was eluted. The concentration of rTES-32 recombinant protein determined for fraction 1 to fraction 10 is shown in table 4. 2. The highest protein concentration was observed in fraction 2 with 1.2 mg/ml. Total protein eluted by this procedure was 3.0 mg/liter of culture.
185 (a) 200 kDa 150 kDa 100 kDa 75 kDa ~ 70 kDa
50 kDa
40 kDa
30 kDa 25 kDa
M 1 2 3 4 5 6 7 8 9 10
(b)
200 kDa 150 kDa 100 kDa 75 kDa ~ 64 kDa
50 kDa
40 kDa
30 kDa 25 kDa
M 1 2 3 4 5 6 7 8 9 10
Figure 4.6 SDS-PAGE analyses of rTES-26 and rTES-32 GST fusion protein fractions
(a) rTES-26; (b) rTES-32
Arrow shows the expressed recombinant proteins.
Lane M : Protein marker (Vivantis, USA)
Lane 1- 10 : Protein fractions 1 to 10
186 Table 4.2 Concentrations of purified rTES-26 and rTES-32 recombinant proteins
Eluted Protein Protein fractions concentration of concentration of TES-26 (mg/ml) TES-32 (mg/ml) F1 0.7 0.4 F2 1.0 1.2 F3 0.67 0.45 F4 0.66 0.5 F5 0.46 0.2 F6 0.45 0.2 F7 0.3 0.1 F8 0.19 0.1 F9 0.1 0.1 F10 0.0 0.0
Total protein yield 4.0 mg/L 3.0 mg/L (in 1 litre of culture medium)
Total protein yield (in 1 litre of culture broth): protein concentration of pooled fractions x total volume of pooled fractions litre of culture
For rTES-26; 0.8 mg/ml x 3.5 ml = 4.0 mg/L 0.5 L
For rTES-32; 0.5 mg/ml x 3 ml = 3.0 mg/L 0.5 L
187 4.3.3 Small-scale optimization of proteolytic cleavage
Figure 4.7 shows the SDS-PAGE profiles of GST cleavage from rTES-26 recombinant protein. The enzyme : target protein ratio of 1:100 and incubation time of 8 hours was considered optimum conditions. Figure 4.8 shows the SDS-PAGE profiles of GST cleavage from rTES-32 recombinant protein. The enzyme : target protein ratio of 1:50 and incubation time of 16 hours was considered optimum conditions. These conditions were chosen because only lower band (size of the proteins without GST tag) appeared while the upper band (size of the GST tagged protein) was completely absent, thus indicating that rTES-26 and rTES-32 recombinant proteins were completely cleaved from the GST tag.
188 (A) 2 hours (B) 4 hours 116.0 Da 116.0 Da 66.2 kDa 66.2 kDa
45.0 kDa 45.0 kDa 35.0 kDa 35.0 kDa
25.0 kDa 25.0 kDa
18.4 kDa 18.4 kDa
M 1 2 3 4 5 6 M 1 2 3 4 5 6
(C) 8 hours (D) 16 hours
116.0 Da 116.0 Da 66.2 kDa 66.2 kDa
45.0 kDa 45.0 kDa
35.0 kDa 35.0 kDa
25.0 kDa 25.0 kDa
18.4 kDa 18.4 kDa
M 1 2 3 4 5 6 M 1 2 3 4 5 6
Figure 4.7 SDS-PAGE analysis of rTES-26 protein after cleavage at different enzyme : protein ratio and incubation times Lane M : Protein marker (Fermentas, USA); Lane 1 : Uncleaved recombinant protein; Lane 2-4 : Ratio 1:20, 1:50 and 1:100, respectively Lane 5 : Positive control; Lane 6 : Negative control
189 (A) 2 hours (B) 4 hours 116.0 Da 66.2 kDa
45.0 kDa
35.0 kDa
25.0 kDa
18.4 kDa 14.4 kDa
6 5 4 3 2 1 M M 1 2 3 4 5 6
190 (C) 8 hours (D) 16 hours
116.0 Da 116.0 Da 66.2 kDa 66.2 kDa
45.0 kDa 45.0 kDa 35.0 kDa 35.0 kDa
25.0 kDa 25.0 kDa
18.4 kDa 18.4 kDa 14.4 kDa 14.4 kDa
M 1 2 3 4 5 6 6 5 4 3 2 1 M
Figure 4.8 SDS-PAGE analysis of rTES-32 protein after cleavage at different enzyme : protein ratio and incubation times Lane M : Protein marker (Fermentas, USA); Lane 1 : Uncleaved recombinant protein; Lane 2-4 : Ratio 1:20, 1:50 and 1:100, respectively Lane 5 : Positive control; Lane 6 : Negative control
191 4.3.4 Determination of immunoreactivity of the purified and cleaved recombinant
proteins by Western blot technique
The Western blot analysis of rTES-120 antigen is shown in figure 4.9. The purified recombinant antigen (target band at approximately 31 kDa) reacted strongly only with sera from patients infected with Toxocara tested against IgG4 antibodies (strips 1 - 3). The recombinant antigen did not cross-react with sera from healthy normals (strips 4 and 5) and sera from patients infected with STH (strips 6 and 7) since no band was detected at the expected size. This study confirmed that rTES-120 recombinant protein is immunologically reactive and specific.
The Western blot analysis of cleaved rTES-26 recombinant protein is shown in figure 4.10. The result showed that no band was detected at 70 kDa (the size of the vector- encoded fusion protein) when probed with mouse monoclonal anti-GST antibody (lane 8).
This confirmed that the rTES-26 GST fusion protein was completely cleaved. The result also demonstrated that the cleaved rTES-26 recombinant protein was antigenic with all the
Toxocara infected patients sera (strips 1 - 3) with the presence of the band at approximately
33 kDa. No reactivity was observed with sera from healthy individuals (strips 4 - 5) and patients infected with STH (strips 6 - 7).
Figure 4.11 shows that the Western blot result of cleaved rTES-32 recombinant protein showed that no band was detected at 64 kDa (the size of the vector-encoded fusion protein) when probed with mouse monoclonal anti-GST antibody (lane 8). This confirmed that the rTES-32 GST fusion protein was completely cleaved. The cleaved rTES-32 recombinant protein was antigenic with all the Toxocara infected patients sera (strips 1- 3) shown by the presence of the band at approximately 31 kDa. No reactivity was observed with sera from healthy individuals (strips 4 - 5) and patients infected with STH (strips 6 - 7).
This part of the study further confirmed that the rTES-26 and rTES-32 recombinant proteins were successfully digested and immunologically reactive and specific.
192 116.0 Da 66.2 kDa 45.0 kDa
35.0 kDa ~ 31 kDa 25.0 kDa
18.4 kDa
14.4 kDa
M 1 2 3 4 5 6 7 8
Figure 4.9 Western blot analysis showing the immunogenicity and specificity of the purified rTES-120 recombinant protein
Arrow shows the expressed recombinant protein.
Lane M : Protein molecular weight marker (Fermentas, USA) Lane 1 : Serum sample from patient infected with toxocariasis (T5) Lane 2 : Serum sample from patient infected with toxocariasis (T7) Lane 3 : Serum sample from patient infected with toxocariasis (T15) Lane 4 : Serum sample from healthy blood donor (PK1) Lane 5 : Serum sample from healthy blood donor (PK2) Lane 6 : Serum sample from patient infected with T. trichiura (Tri 1) Lane 7 : Serum sample from patient infected with A. lumbricoides (Asc 1) Lane 8 : Mouse monoclonal anti-histidine-HRP (SantaCruz, USA)
193
66 kDa
45 kDa
~ 33 kDa 30 kDa
20.1 kDa
14.4 kDa M 1 2 3 4 5 6 7 8
Figure 4.10 Western blot analysis showing the immunogenicity and specificity of cleaved rTES- 26 recombinant protein
Arrow shows the expressed recombinant protein.
Lane M : Low molecular weight marker (Amersham BioSciences, Sweden) Lane 1 : Serum sample from patient infected with toxocariasis (T5) Lane 2 : Serum sample from patient infected with toxocariasis (T7) Lane 3 : Serum sample from patient infected with toxocariasis (T15) Lane 4 : Serum sample from healthy blood donor (PK1) Lane 5 : Serum sample from healthy blood donor (PK2) Lane 6 : Serum sample from patient infected with T. trichiura (Tri 1) Lane 7 : Serum sample from patient infected with A. lumbricoides (Asc 1) Lane 8 : Mouse monoclonal anti-GST antibody (Novagen, Germany)
194 97 kDa 66 kDa
45 kDa
30 kDa ~ 30 kDa
20.1 kDa
14.4 kDa
M 1 2 3 4 5 6 7 8
Figure 4.11 Western blot analysis showing the immunogenicity and specificity of cleaved rTES-32 recombinant protein
Arrow shows the expressed recombinant protein.
Lane M : Low molecular weight marker (Amersham BioSciences, Sweden) Lane 1 : Serum sample from patient infected with toxocariasis (T5) Lane 2 : Serum sample from patient infected with toxocariasis (T7) Lane 3 : Serum sample from patient infected with toxocariasis (T15) Lane 4 : Serum sample from healthy blood donor (PK1) Lane 5 : Serum sample from healthy blood donor (PK2) Lane 6 : Serum sample from patient infected with T. trichiura (Tri 1) Lane 7 : Serum sample from patient infected with A. lumbricoides (Asc 1) Lane 8 : Mouse monoclonal anti-GST antibody (Novagen, Germany)
195 4.4 Discussion
This chapter described the techniques used to purify the recombinant proteins rTES-26, rTES-32 and rTES-120. The methods chosen for the recombinant protein purifications depended mainly on its location and form within the cell. The aim of the study was to produce recombinant proteins that can be employed in developing an ELISA for the detection of Toxocara infections. Hence, the most important criterion was to produce purified recombinant proteins in sufficient quantities. In addition the purities of the recombinant antigens should produce sensitive and specific reactivities (by Western blots) to
Toxocara and non-toxocara sera samples respectively.
Purification under native conditions was used to purify rTES-26, rTES-32 and rTES-
120 recombinant proteins since they were abundantly expressed in soluble form in the cytoplasm of the E. coli. Insoluble proteins which are present as inclusion bodies are devoid of biological activity and need elaborate solubilization, refolding and purification procedures to recover functionally active product. Protein solubilization using high concentrations of chaotropic reagents results in the loss of secondary structure. This condition combined with the interaction among the denatured protein molecules usually result in protein aggregation which lead to poor recovery of bioactive proteins from the inclusion bodies. Although bioactive proteins are not required for use as diagnostic reagents, soluble proteins are much more preferred (to insoluble proteins) due to much greater ease of production (Cabrita et al.,
2004). The protocol involved in solubilization and refolding of the proteins are tedious and usually results in poor recovery (Singh & Panda, 2005). In addition soluble proteins also have much less risk of loss of immunogenicity upon long term storage as compared to proteins stored in chaotropic reagents. Thus, a lot of effort was made to try to produce the soluble form of the protein. At the beginning of the purification study, the rTES-120 recombinant protein could only be produced in an insoluble form. This may be due to the overexpression of the recombinant protein. The expression strategy was modified in two of the steps namely i.e. the overnight culture was incubated at 30ºC, 180 rpm (instead of 37ºC,
200 rpm) and the IPTG induction was performed at O.Ds 0.5 (instead of 0.8). These
196 conditions resulted in decrease of the protein synthesis rate. Using these strategies, we were able to produce rTES-120 recombinant protein in soluble form. As mentioned previously the rTES-120 recombinant protein produced by other researchers was insoluble (Fong et al.,
2003; 2004).
In this study, purification of rTES-120 histidine-tagged protein is based on the principles of immobilized metal chelate affinity chromatography (IMAC) using precharged
Ni SepharoseTM High Performance (Amersham BioSciences, Sweden). IMAC technology has become popular and is very useful technique to purify the histidine-tagged proteins because it is both highly effective and relatively insensitive to ionic strength, chaotropes and detergent (Hochuli et al., 1988). Ni SepharoseTM High Performance consists of highly cross- linked agarose beads to which a chelating group has been immobilized. The metal ion nickel
(Ni2+) has been coupled to the chelating matrix. The optimization of rTES-120 histidine- tagged purification was carried out using FPLC automated system using Ni SepharoseTM resin. FPLC method is capable to purify a large amount of protein in a shorter time than manual purification method. Other resins that have been extensively used to purify histidine- tagged proteins are Ni-NTA (nickel-base resin) and HisPur resin (cobalt-based resins).
Polyhistidine tags are often used for affinity purification of polyhistidine-tagged recombinant proteins that are expressed in E. coli or other prokaryotic expression systems since they generally do not destroy protein functions and allow affinity purification to be carried out under gentle conditions. The histidine tag is the most widely used affinity tag due to its small size, strong metal ion binding and ability to bind under native as well as denaturing conditions (Baneyx, 1999). Affinity purification using a polyhistidine tag usually results in relatively pure protein when the recombinant protein was expressed in prokaryotic host organism.
The 6xHis-tagged proteins have a higher affinity for the Ni SepharoseTM resin than background proteins. Consequently, very few non-tagged proteins will be retained on the resin if nearly all available binding sites are occupied by the tagged protein. If too much matrix was used, other proteins may nonspecifically bind to unoccupied sites and elute as
197 contaminants. The potential for non-tagged proteins to interact with the resin is usually higher under native than under denaturing conditions. Proteins that copurify because they are linked to the 6xHis-tagged protein, proteins that associate non-specifically with the tagged protein, and nucleic acids that associate with the tagged protein can appear as contaminants in the eluate. However, the non-specific binding was minimized by including a low concentration of imidazole (10 mM) in the lysis buffer.
Phosphate buffers were used for purification of rTES-120 histidine-tagged protein due to their low tendency to strip metal ions from IMAC matrices. The protein was washed using a continuous gradient of increasing imidazole concentrations from 20 mM to 40 mM.
The purity of the recombinant protein was much improved by gradient washing as opposed to using one concentration of imidazole. Moreover recently, this technique has been successfully used to produce rTES-30USM antigen in our laboratory (Norhaida et al., 2008).
After unbound proteins are washed away, the target protein was eluted with imidazole. The imidazole concentrations of the wash and elution buffers under native conditions were adjusted to minimize copurification of nonspecifically bound proteins. If the imidazole concentration in the wash buffer is higher than 100 – 250 mM, the 6xHis-tagged proteins will also dissociate because they can no longer compete for binding sites to the nickel ions on the resin. Imidazole is mildest as elution buffer and is recommended under native conditions, whereas the protein may be damaged by eluting using pH reduction.
Some proteins may exhibit higher solubility when the cells are lysed in a buffer containing salt. In this study, the salt concentrations used during binding and washing steps was optimized (0.3 and 0.5 mM) to get the optimum conditions for purification. The results showed that the yield of TES-120 recombinant protein was higher when using buffers containing 0.3 M NaCl (1.5 mg/L) as compared to buffers containing 0.5 M NaCl (1.25 mg/
L). However, the SDS-PAGE results revealed that purity of the antigen was better in buffers containing 0.5 M NaCl (figures 4.5c and d) compared to 0.3 M NaCl (figures 4.5a and b).
Sufficient ionic strength in the purification buffers help in preventing non-specific interactions between proteins and the charged resin. This was shown by the reduction of the
198 extra bands (~ 45 kDa) in buffers containing 0.5 M NaCl starting from fraction 5 (figures
4.5c and d). The increase of salt in the buffer increased the ionic strength of the buffer therefore increasing the purity (Hochuli et al., 1988). However, there was no significant difference in 5 and 10 column volumes for both buffers. Therefore, purification using buffers containing 0.5 M NaCl at 5 CV were chosen as the optimum conditions.
The SDS-PAGE (Fig 4.5) showed that there were impurities in rTES-120 protein.
This is because the amino acid histidine was also present as part of the protein of the bacterial host cells, therefore to achieve very high purity in histidine-tagged protein (by more stringent washing steps) is usually difficult without significantly compromising the quantity of the recombinant protein. In this study, the immunogenicity of the recombinant protein was clearly not affected by these impurities since only one protein band was detected during the
Western blot analysis with serum samples from Toxocara infection, and no reactivity observed when probed with other serum samples, thus no attempt was made to achieve higher purity so as not to affect the yield of the recombinant protein. For future upscaling of the production of the recombinant protein, the purification strategy will be further fine-tuned to achieve high purity and yield.
It is well known that histidine tag is a very small peptide (of only about 0.8 kDa), is not immunogenic and its neutral charge at physiological pH means that it rarely interferes with the protein structures as well as functions and less disruptive to the properties of the proteins on which they are attached (Hochuli et al., 1988). Therefore, in this study the tag was not removed.
Fusion of target proteins with a highly soluble protein such as GST has been shown to increase the soluble fraction of the target proteins (Smith & Johnson, 1988). Thus GST was used as fusion partner of rTES-26 and rTES-32 fusion proteins. For purification of the proteins, GST BindTM purification system was employed. This is based on the widely recognized affinity of glutathione S-transferase (GST TagTM) fusion proteins for immobilized glutathione. GST Bind Resin is a cross-linked agarose matrix to which reduced glutathione is covalently attached via a sulfide linkage using an 11-atom spacer arm to ensure a high
199 binding capacity with yields of 5 to 8 mg/ml settled resin for rapid and easy purification to near homogeneity in single-step of recombinant GST fusion proteins containing the 220 amino acid GST domain (Novagen, Germany). In rTES-26 and rTES-32 recombinant proteins, the GST tag is fused to the N-terminal end of these proteins. The fusion protein containing the GST Tag expressed with the pET-42 vector was then detected with mouse monoclonal anti-GST antibody.
The purification of rTES-26 and rTES-32 GST fusion proteins was performed with manual method using batch/gravity flow. In this procedure, the recombinant proteins were allowed to bind to the resin in solution for a certain period of time before the protein-resin mixture was applied to the column. This method provided efficient binding of the recombinant protein to the matrices. The purification of these GST fusion proteins were not performed by FPLC. This was because the automated method requires a larger amount of washing and elution buffers. In this study, the elution buffer was only commercially available and thus could only be prepared in small amounts.
Since GST tag is a large molecule (of about 26 kDa), and may affect the immunogenicity of the protein, thus it was necessary to remove it prior to employing it in immunoassays. The pET vector (Novagen, Germany) includes a Factor Xa domain for cleavage of the GST tag during protein purification. In pET-42 vectors, the Factor Xa (Ile-
Glu-Gly-Arg) proteolytic cleavage site has been engineered to allow 100% complete removal of vector encoded sequences and the generation of native proteins with their authentic N-terminal residues. In this study, the restriction grade site-specific proteases,
Factor Xa was used for removal of GST tag in the rTES-26 and rTES-32 GST-tagged fusion proteins. The Factor Xa Cleavage Capture Kit (Novagen, Germany) is designed for highly specific cleavage of GST-tagged fusion proteins followed by convenient affinity-based capture and removal of Factor Xa enzyme. After cleavage of the target protein, Factor Xa is removed with greater than 95% efficiency from the reaction by affinity capture on XarrestTM
Agarose. XarrestTM Agarose is used for the quantitative removal of Factor Xa, following cleavage of fusion proteins. Following capture of Factor Xa, the agarose was removed by
200 spin-filtration. The performance of the cleavage reaction was monitored by SDS-PAGE analysis.
The optimal cleavage conditions were determined individually for each protein to be cleaved. Optimization of the cleavage conditions was performed in small-scale reactions.
Optimization of Factor Xa Protease concentration and incubation times (2 – 16 hours) was performed because excess of Factor Xa Protease may result in non-specific proteolysis at secondary sites. Therefore, optimal enzyme specificity is achieved using the lowest amount of protease necessary to achieve complete cleavage. The Factor Xa Protease activity is sensitive to the concentration of protein to be cleaved. Therefore, a minimum of 10 µg protein per 50 µl reaction which is recommended by manufacturer was used in this study
(Novagen, Germany). The ratios of enzyme to target proteins at 1:100 and 1:50 and incubation times 8 and 16 hours were found to provide optimum cleavage conditions for rTES-26 and rTES-32, respectively. When the conditions were optimized, only the lower band (recombinant proteins without GST tag) appeared while the upper bands (recombinant proteins with the GST tag) was absent (figures 4.7 and 4.8), thus signaling that recombinant protein was completely cleaved from the GST tag.
The molecular weight of GST-rTES-26, GST-rTES-32 and His-rTES-120 recombinant proteins predicted using Vector NTi were 72, 64 and 31 kDa, respectively.
These values corresponded with the size of the proteins observed using SDS-PAGE/Western blot. Other bands located below the TES recombinant proteins band were also observed
(figures 4.5 – 4.6). These bands were probably degraded products of TES recombinant proteins due to partial degradation of fusion proteins by proteases, or denaturation and co- purification of host proteins with the recombinant protein due to excessive cell breakage.
Over-sonication may have denatured the fusion protein and preventing it from binding to the resin. Therefore, in this study French press which is milder than sonication was used during cell lysis to minimize the denaturation. The addition of protease inhibitor and lysozyme prior to cell lysis are important in this regard. Lysozyme avoids the cataclysmic lysis that can release membrane proteins and other molecules that cause aggregation of the polyhistidine-
201 tagged or GST fusion protein. E. coli BL21 is a protease-deficient strain specifically selected to give high efficiency transformation and high level of expression of GST fusion proteins. If one uses E. coli strains that are not protease-deficient, this may result in much greater proteolysis of the fusion protein ((Baneyx, 1999).
GST tag system was used for rTES-26 and rTES-32 proteins since it was reported to produce high purity protein, since the GST molecule is not present in the bacterial host.
However there were a lot of problems that arose during the purification of these proteins especially those related to the stability prior to cleavage of the tag. In addition, the high cost of GST resin and the enzyme used for cleavage was also prohibitive. Thus, the cloning of the subsequent recombinant protein (rTES-120) was performed using a vector with histidine tag.
Besides the advantage of obtaining highly purified proteins with GST system, there are a few important things that need to be considered. Unlike histidine tag, the large size of
GST results in a higher potential for degradation by proteases than other smaller tags. This results in multiple bands due to partial degradation of fusion proteins (Baneyx, 1999).
Therefore, performing rTES-26 and rTES-32 GST-tagged proteins purification as quickly as possible under nondegrading conditions was necessary in order to minimize sample loss.
Glutathione-resin based purifications require that the GST domain is soluble and properly folded. GST loses its ability to bind glutathione resin when denatured. The GST fusion proteins should not be kept frozen before the removal of GST tag. Thus, the cleavage of GST tag has to be performed as soon as possible to prevent degradation after purification
((Baneyx, 1999).
The Western blot analysis of rTES-26, rTES-32 and rTES-120 further confirmed the presence of rTES-120 His-tagged protein (figure 4.11) and no band was detected in rTES-26 and rTES-32 proteins for the GST detection (figures 4.9 and 4.10, respectively). This showed that the rTES-26 and rTES-32 were completely cleaved from the vector-encoded sequences. The results also shown that all purified recombinant antigens strongly reacted only with sera from patients infected with Toxocara when tested using monoclonal mouse anti-human IgG4 antibodies. These recombinant antigens did not cross-react with sera from
202 healthy normals and sera from patients infected with STH. In this chapter, only IgG4 was employed because past experiences showed that IgG4 assay is specific for detection of toxocariasis (Rahmah et al., 2005; Norhaida et al., 2008).
This study is the first report describing the use of GST tag in the expression and purification of rTES-26 and rTES-32 recombinant proteins. The rTES-26, rTES-32 and rTES-120 recombinant proteins were successfully purified to sufficient purity with high protein concentrations and were proved to be immunologically reactive and specific.
203 CHAPTER 5
DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
USING rTES-26, rTES-32 AND rTES-120 RECOMBINANT ANTIGENS
5.1 Introduction
The most effective laboratory diagnostic approach for confirming Toxocara as the aetiological agent of disease in clinically suspected cases is by serology (Smith, 1998;
Magnaval et al., 2001). IgG-ELISA has been shown to be of value in the diagnosis of
Toxocara infections in humans. TES antigens from T. canis second-stage larvae have been widely used to coat ELISA microtiter plates. However, the TES antigens are cross-reactive against serum samples from patients with a variety of helminth infections (Lynch et al.,
1988; Magnaval et al., 1991; Jacquier et al., 1991; Gillespie et al., 1993; Yamasaki et al.,
1998). Therefore, to overcome such cross-reactivity problems, TES recombinant antigens which are more specific than native TES have been produced in this study (Yamasaki et al.,
2000).
In the previous chapter of this thesis, rTES-26, rTES-32 and rTES-120 recombinant antigens were demonstrated by Western blot to be sensitive and specific for the detection of
Toxocara-specific IgG4 antibodies. In this chapter, an ELISA based on these recombinant antigens was developed and evaluations of the diagnostic value of the assay were performed using monoclonal mouse anti-human IgG subclasses (IgG1, IgG2, IgG3, IgG4), IgE and
IgM. Subsequently, evaluations were carried out using a panel of sera comprising Toxocara infections, other helminth related infections and healthy individuals in order to validate the sensitivities and specificities of the assays. In this chapter, rTES-30USM previously developed in our laboratory (Norhaida et al., 2008) was also included in one part of the analysis.
204 5.2 Materials and methodology
5.2.1 Total IgE-ELISA
Total IgE ELISA (Total IgE ELISA kit, IBL Hamburg, Germany) was performed for the quantitative determination of total IgE in human serum of Toxocara patients, other helminthic infections and healthy normals. This assay was carried out in order to further support the diagnosis of Toxocara infections since total serum IgE levels in toxocariasis patients were often reported to be elevated (Figuieredo et al., 2005).
This assay is a solid phase enzyme-linked immunosorbent assay based on the sandwich principle. The monoclonal mouse-anti-human IgE antibody was bound on the surface of the microtiter strips. All reagents (ready-to-use) and serum samples were allowed to equilibrate at room temperature before use. Each standard containing different concentrations (in IU/ml) of human IgE diluted with horse serum (A: 0; B: 5; C: 25; D: 100;
E: 250; F: 1000) and serum samples (10 µl each) were added into the appropriate wells
(starting with well 2). Well 1 was reserved for the substrate blank. Enzyme conjugate containing goat anti-human IgE-HRP (200 µl) was added into each well (except well 1) and thoroughly mixed for 15 seconds. After incubation for 30 minutes at room temperature, the wells were washed three times with 300 µl of wash buffer using a microtitre plate washer
(Columbus/Columbus Pro Washer, TECAN, Austria). Subsequently, TMB substrate solution
(100 µl) was added into each well (including well 1). After incubation for 15 minutes at room temperature in the dark, the TMB stop solution containing 1 M sulphuric acid (100 µl) was added into each well. Finally, the results were read at 450 nm (against the substrate blank) with an automatic ELISA plate reader (TECAN Sunrise, Sweden). The normal value of total IgE in humans should be less than 100 IU/ml.
5.2.2 Optimization of various parameters in rTES ELISA
Prior to screening of the serum samples, some important parameters were optimized in order to get the optimum conditions for each assay. These include optimization on the best
205 concentration of the antigen to be used for coating onto ELISA microtiter wells and the optimum dilutions of primary and secondary antibodies to be used in each assay.
5.2.3.1 Concentration of rTES recombinant antigens
A checkerboard titration was performed to optimize the concentration of recombinant antigens used for coating of microtiter plate. The optimal concentration of antigens needed to saturate available sites on a microtiter plate was evaluated using a range of antigen concentrations namely 2.5, 5, 10, 20 and 40 µg/ml in coating buffer, pH 9.6. The coated plate was then incubated at 4ºC overnight in a humid chamber to allow the recombinant antigens to be well-adsorbed onto microtiter plate.
5.2.3.2 Dilutions of primary antibodies
The primary antibody used in this study was serum sample. The antibodies in the serum samples recognized the specific epitopes on the coated antigens. A checkerboard titration was performed to optimize the dilutions of the primary antibody for IgG4-assay. Dilutions of
1:50 and 1:100 were prepared using PBS, pH 7.2 as the diluent. However, only the dilution of 1:50 was used for other assays since it was shown to give the best discrimination between positive and negative samples in IgG4-ELISA assay.
5.2.3.3 Dilutions of secondary antibodies
The secondary antibodies used in this study were monoclonal mouse anti-human IgG1-4 subclasses, IgE and IgM conjugated with horseradish-peroxidase (HRP) enzyme, from
Zymed (USA) except for IgG2 (Immulon, USA). The recommended working dilutions given by the manufacturer are between 1:500 and 1:2000 (for IgG2-4 and IgE), 1:500 and 1:1000
(for IgM) and 1:10,000 to 1:15,000 (for IgG1). Excessive concentration of secondary antibodies will elevate background noise. Thus, checkerboard titrations were performed in order to obtain optimal dilutions for each of the secondary antibodies. Thereafter, dilutions
206 of 1:500, 1:1000 and 1:2000 (for IgG2-4 and IgE), 1:500 and 1:1000 (for IgM) and 1:10,000 and 1:15,000 (for IgG1) were prepared using PBS, pH 7.2 as the diluent.
5.2.3 Indirect enzyme-linked immunosorbent assay (ELISA)
Each well of the 96-well flat-bottomed microtiter plate (NUNC ImmunoTM Maxisorp,
Denmark) was coated with 100 µl of each recombinant antigen at optimum concentrations for each antigen in coating buffer (pH 9.6). The plate was then covered with aluminium foil and placed in a humid chamber by placing wet tissue papers at the bottom of the chamber, then incubating at 4ºC, overnight.
All washing steps were performed using a microtitre plate washer
(Columbus/Columbus Pro Washer, TECAN, Austria) 5 times for 5 minutes each. The plate was washed with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T), pH 7.2 to remove any unadsorbed antigen. Then, each well was blocked with 200 µl of 1.0% blocking reagent (Roche Diagnostics, Germany) for 1 hour at 37ºC. The plate was again washed as previously described, followed by addition of the diluted serum (100 µl/well diluted in PBS, pH 7.2) to each well and incubated at 37ºC for 2 hours. After washing off the excess sera, monoclonal mouse anti-human IgG1-4 subclasses / IgE / IgM conjugated to HRP (Zymed,
USA) was added at the optimum dilutions for each of the recombinant antigen (100 µl/well diluted in PBS, pH 7.2) and incubated at 37ºC for 30 minutes. Following a final washing step, ABTS substrate (Roche Diagnostics, Germany) was added and the results were read after 30 minutes with 410 nm test filter (495 nm reference filter) with an automatic ELISA plate reader (TECAN Sunrise, Sweden). Each test plate had PBS (as control blank), positive and negative serum controls. All sera were tested in duplicate and results were expressed as optical density (OD) values.
An IgG4-ELISA assay using rTES-30USM was carried out as previously described
(Norhaida et al., 2008). The protocol was the same as described above with some modifications i.e. recombinant antigen was used at 20 µg/ml, 2.0% blocking reagent, serum
207 samples (1:100, diluted in 1.0% blocking reagent) and monoclonal mouse anti-human IgG4-
HRP at 1:500 (diluted in 1.0% blocking reagent).
5.2.4 Determination of positive cut-off optical density value (COV)
An O.D reading of 0.200 was used as the cut-off value in all assays to discriminate between positive and negative. This COV was based on mean O.D reading plus three standard deviations (SD) of 30 serum samples from healthy individuals. The O.D readings were substracted from the O.D of PBS (as blank) and O.D readings of equal and/or greater than
0.200 were interpreted as positive cases.
In order to assess the sensitivity of the ELISA using antibodies to IgG subclasses
(IgG1 – 3), IgE and IgM, the assay was carried out on 20 serum samples with Toxocara infections, while specificity was assessed using 65 serum samples from patients with different helminthic infections as well as healthy normals. The reason for the reduced numbers of samples was due to insufficient volumes of available sera.
The sensitivity of a test is the probability that the test is positive when tested with a group of patients with the disease. The formula for sensitivity (Sn) is:
Sn = [TP / (TP + FN)] x 100
The specificity of a test is the probability that the test is negative among patients who do not have the disease. The formula for specificity (Sp) is:
Sp = [TN / (TN + FP)] x 100
An accuracy of a test is the proportion of true results (both true positive and true negative) in the population. The formula for accuracy is:
208 Accuracy = [(TP + TN)/(TP + FN + FP + TN)] x 100
The positive predictive value of a test is the probability that the patient has the disease when restricted to those patients who test positive. The formula for PPV is:
PPV = [TP / (TP + FP)] x 100
The negative predictive value of a test is the probability that the patient will not have the disease when restricted to those patients who test negative. The formula for NPV is: NPV =
[TN / (TN + FN)] x 100 where TP (true positive: an individual that is truly positive for toxocariasis), TN (true negative: an individual that is truly negative for toxocariasis), FP (false positive: an individual that is truly disease-free, but which a diagnostic test classifies as positive for toxocariasis), FN (false negative: an individual that is truly positive for toxocariasis, but which a diagnostic test classifies as disease-free).
5.2.5 Method validation
5.2.5.1 Determination of sensitivity, specificity, accuracy, positive predictive value
(PPV) and negative predictive value (NPV) of IgG4-ELISA
The analysis of previously produced rTES-30USM (Norhaida et al., 2008) was included in this part of the study (together with rTES-26, rTES-32 and rTES-120) since it (rTES-
30USM) was shown to be highly sensitive and specific for detection of toxocariasis. The diagnostic utility of each IgG4-ELISA developed was evaluated by using 30 serum samples with Toxocara infection and 112 serum samples from patients with different parasitic infections (STH, amoebiasis, B. malayi microfilaremic, toxoplasmosis, strongyloidiasis, gnathostomiasis) and 100 serum samples from healthy individuals.
5.2.5.2 Statistical analysis
All statistical significance of the values obtained was expressed at 95% confidence level.
These analyses were performed using the Statistical Package for Social Science (SPSS) version 12.0. The positivity rate in total serum IgE-ELISA assay was compared between
209 Toxocara infections (25 serum samples) and each of the following groups of serum samples, namely STH (28), B. malayi microfilaremics (15), amoebiasis (12), toxoplasmosis (12), strongyloidiasis (5) and healthy normals (68) by Chi-square to determine if significance difference exists between the groups.
The Chi-square analysis was also employed to determine whether the observed difference in sensitivity rate between rTES-26/rTES-32 (80.0%) and rTES-30USM/rTES-
120 (93.3%) was statistically significant. One-way ANOVA analysis was used to compare the degree of reactivities of these recombinant antigens by comparing the mean O.Ds obtained by each of the recombinant antigens using 30 sera from toxocariasis patients. The specificity differences obtained between rTES-26, rTES-32, rTES-120 and rTES-30USM were analyzed by Chi-Square test in two ways i.e. by including 30 sera from healthy normal group and by excluding the healthy group. The p value of < 0.05 was considered statistically significant.
5.4 Results
5.3.1 Determination of total IgE in sera from toxocariasis patients, various
helminthic infections and healthy normals
The results of the total IgE from various categories of serum samples are shown in table 5.1.
Out of 25 Toxocara patients tested, 21 (84.0%) have high level of total serum IgE. More than 50% of sera from patients infected with STH, B. malayi microfilaremics and amoebiasis also showed an increased level of total IgE. Some of the sera from toxoplasmosis and strongyloidiasis as well as healthy individuals also showed an increased level of total IgE.
Table 5.2 shows the summary of the results of the screening of total IgE in sera from toxocariasis patients, various parasitic infections and healthy normals.
The Chi-square analysis of positivity in total serum IgE showed that Toxocara patients had significantly greater number of positive individuals as compared to STH
(p<0.001), B. malayi microfilaremics (p<0.001), amoebiasis (p<0.001), toxoplasmosis
(p<0.001) and healthy normals (p<0.001).
210 Table 5.1 Results of the screening of various categories of serum samples using Total IgE-ELISA (Total IgE-ELISA kit, IBL Hamburg, Germany)
No Serum Code OD Concentration Interpretation 1. T1 0.383 59.52 - 2. T2 1.076 245.20 + 3. T3 2.408 1025.00 + 4. T4 2.413 1028.40 + 5. T5 2.400 1012.20 + 6. T6 2.001 998.25 + 7. T7 0.963 200.59 + 8. T8 0.411 80.16 - 9. T9 2.269 1005.30 + 10. T10 1.415 571.28 + 11. T12 2.247 1000.00 + 12. T15 1.218 373.49 + 13. UMT#3 2.256 1003.55 + 14. UMT#6 2.008 999.50 + 15. UMT#9 1.097 250.20 + 16. MBDR60 1.725 882.53 + 17. MBRD62 1.832 989.96 + 18. MBDR65 0.193 4.63 - 19. MBDR66 1.672 829.32 + 20. MBDR68 1.926 998.64 + 21. T16 1.04 232.52 + 22. T17 1.664 821.28 + 23. 96.41358.M 0.178 4.00 - 24. 96.28371.H 1.409 553.19 + 25. 98.29664.V 0.990 223.72 + 26. PK1 0.137 4.45 - 27. PK2 0.133 4.44 - 28. PK3 0.079 2.16 - 29. PK4 1.016 225.46 + 30. PK5 0.190 4.63 - 31. PK6 0.086 2.57 - 32. PK7 0.634 129.65 + 33. PK8 0.457 81.61 - 34. PK9 0.639 130.58 + 35. PK10 0.212 21.35 - 36. PK11 0.333 47.07 - 37. PK12 1.024 240.99 + 38. PK13 0.131 4.36 - 39. PK14 0.785 167.61 + 40. PK15 0.436 73.53 - 41. PK16 1.17 328.85 + 42. PK17 0.122 4.06 - 43. PK18 1.713 838.55 +
211 Table 5.1(cont.) Results of the screening of various categories of serum samples using Total IgE-ELISA (Total IgE-ELISA kit, IBL Hamburg, Germany)
44. PK19 0.007 0.20134 - 45. PK20 2.116 1000.28 + 46. PK21 0.270 30.45 - 47. PK22 0.005 0.00 - 48. PK23 0.347 50.43 - 49. PK24 0.105 3.49 - 50. PK25 0.766 162.41 + 51. PK26 0.171 9.24 - 52. PK27 1.299 449.94 + 53. PK28 0.01 0.00 - 54. PK29 0.216 18.33 - 55. PK30 1.599 731.54 + 56. PK31 0.002 0.03 - 57. PK32 1.214 370.15 + 58. PK33 0.306 39.76 - 59. PK34 0.036 1.17 - 60. PK35 0.189 12.88 - 61. PK36 0.266 29.41 - 62. PK37 0.100 3.32 - 63. PK38 0.800 171.71 + 64. PK39 2.041 999.65 + 65. PK40 0.471 82.612 - 66. PK41 1.091 254.69 + 67. PK42 2.044 999.66 + 68. PK43 0.085 2.82 - 69. PK44 0.77 163.50 + 70. PK45 0.106 3.52 - 71. PK46 0.148 4.93 - 72. PK47 0.643 128.74 + 73. PK48 1.061 243.16 + 74. PK49 0.06 1.98 - 75. PK50 0.162 7.42 - 76. PK51 0.238 22.78 - 77. PK52 0.181 11.26 - 78. PK53 1.495 633.92 + 79. PK54 0.470 82.35 - 80. PK55 0.382 59.52 - 81. PK56 0.041 1.34 - 82. PK57 0.134 4.46 - 83. PK58 0.029 0.94 - 84. PK59 0.273 31.23 - 85. PK60 0.334 47.06 - 86. PK61 0.122 4.06 - 87. PK62 0.561 106.30 + 88. PK63 0.388 61.07 -
212 Table 5.1(cont.) Results of the screening of various categories of serum samples using Total IgE-ELISA (Total IgE-ELISA kit, IBL Hamburg, Germany)
89. D191 0.021 0.85 - 90. D133 0.112 3.99 - 91. D135 0.012 0.59 - 92. D164 0.117 4.02 - 93. D201 0.014 0.63 - 94. Tri/2 1.999 989.12 + 95. Mix/5 1.533 689.76 + 96. Mix/15 2.025 998.89 + 97. Tri/1 2.200 1000.00 + 98. Tri/5 1.212 370.15 + 99. Tri/6 1.312 385.69 + 100. Tri/10 2.315 1009.88 + 101. Tri/11 2.210 1002.45 + 102. Tri/12 2.511 1050.40 + 103. Asc/1 2.310 1008.20 + 104. Asc/2 0.210 29.65 - 105. Asc/3 2.058 998.90 + 106. Asc/7 0.130 3.598 - 107. Asc/9 2.318 1009.00 + 108. Asc/10 0.230 30.09 - 109. Mix/7 0.125 3.99 - 110. Mix/8 0.510 91.69 - 111. Mix/10 0.525 92.00 - 112. Mix/14 0.499 90.04 - 113. Mix/16 0.485 89.95 - 114. Mix/17 0.251 25.63 - 115. Mix/18 1.250 450.74 + 116. Mix/19 1.018 300.45 + 117. Tri/8 0.001 0.00 - 118. Mix/1 0.009 0.27 - 119. Mix/6 0.007 0.20 - 120. Mix/13 0.010 0.30 - 121. Nec/2 1.774 895.81 + 122. A32 0.946 226.80 + 123. EH59 0.502 88.52 - 124. EH46 0.309 39.29 - 125. EH10 1.906 899.00 + 126. EH47 1.115 346.39 + 127. EH76 1.375 607.43 + 128. EH1 1.585 756.82 + 129. EH14 0.025 1.00 - 130. EH5 2.580 1100.24 + 131. A39 2.218 1003.05 + 132. A49 0.013 0.09 -
213 Table 5.1(cont.) Results of the screening of various categories of serum samples using Total IgE-ELISA (Total IgE-ELISA kit, IBL Hamburg, Germany)
133. EH62 0.150 4.99 - 134. STG 2.1 1.104 266.90 + 135. STG 2.8 1.430 572.90 + 136. STG 3.2 0.476 83.91 - 137. STG 3.5 0.101 3.36 - 138. STG 3.6 1.562 696.81 + 139. Gnat 1 0.242 23.59 - 140. UMT78 0.071 2.73 - 141. UMT70 1.321 538.85 + 142. UMT6 2.211 1001.55 + 143. UMT71 0.008 0.06 - 144. UMT21 0.031 1.22 - 145. UMT83 0.221 19.89 - 146. UMT45 0.011 0.56 - 147. UMT66 0.369 56.14 - 148. UMT35 0.767 162.68 + 149. UMT50 0.057 1.88 - 150. TP1 0.024 1.00 - 151. TG1 1.200 450.35 + 152. SB1 0.339 58.69 - 153. SB6 1.461 635.21 + 154. SB30 0.096 3.00 - 155. SB57 2.106 1000.00 + 156. SB31 1.394 539.11 + 157. SB9 1.216 372.03 + 158. SB21 0.506 91.69 - 159. SB17 1.248 402.07 + 160. SB52 1.892 900.99 + 161. SB26 0.009 0.27 - 162. SB32 0.006 0.17 - 163. SB55 0.031 1.00 - 164. SinSB59 0.570 108.76 + 165. RajWb 1.478 617.96 + 166. MF20 1.529 665.83 +
Note: No. 1 – 25: Toxocariasis; 26 – 93: Healthy normals; 94 – 121: STH; 122 – 133: Amoebiasis; 134 – 138: Strongyloidiasis; 139: Gnathostomiasis; 140 –151: Toxoplasmosis; 152 – 166: B. malayi microfilaremic individuals.
Numbers in bold and highlighted showed high level of total serum IgE (more than 100 IU/ml).
214 Table 5.2 Summary of the results of the screening of total IgE in sera from toxocariasis patients, various parasitic infections and healthy normals (summarized from table 5.1)
Type of serum Total Positive Negative Positivity rate (%) Toxocariasis 25 21 4 84.0 STH 28 15 13 53.6 B. malayi microfilaremics 15 9 6 60.0 Amoebiasis 12 7 5 58.3 Toxoplasmosis 12 4 8 25.0 Strongyloidiasis 5 3 2 60.0 Gnathostomiasis 1 0 1 0 Healthy normals 68 21 47 44.7 Total 166
5.4.2 Optimization of different parameters in rTES ELISA
215 The results of each optimization assay are shown in Tables 5.3 – 5.8, a summary of the optimum conditions for each assay are depicted in Table 5.9. These optimum conditions were chosen because they gave the best discrimination of O.Ds between positive and negative sera. Based on the optimum serum dilution in IgG4-ELISA, the same dilution of serum was employed for other assays (IgG1-IgG3, IgE, IgM), and optimizations of these other assays were limited to concentration of antigens and conjugates. Based on the results obtained, it was deemed not necessary to test with other serum dilutions since either the O.D of the Toxocara serum samples were low and/or the O.D of the non-toxocara serum samples were very high. These optimized parameters were then employed in each assay in order to evaluate its usefulness for the detection of Toxocara infection.
Table 5.3a Optimization of rTES-26 recombinant antigen concentration and serum dilution in IgG4-ELISA
216 Antigen conc. (μg/ml) 5 10 20 40 5 10 20 40 Serum Serum Code dilution 1:50 1:50 1:50 1:50 1:100 1:100 1:100 1:100 Serum description T2 Toxocariasis 0.344 0.717 0.623 0.690 0.198 0.425 0.499 0.415 T5 Toxocariasis 1.212 2.077 2.100 2.035 0.592 0.985 1.025 1.122 T7 Toxocariasis 1.105 1.993 1.903 1.953 0.663 1.256 1.299 1.125 T12 Toxocariasis 2.764 3.490 3.407 3.318 1.119 1.912 2.120 2.098 PK1 Healthy 0.032 0.051 0.084 0.151 0.025 0.028 0.029 0.048 normals PK2 Healthy 0.086 0.095 0.121 0.196 0.050 0.062 0.065 0.066 normals PK3 Healthy 0.035 0.050 0.097 0.150 0.029 0.035 0.048 0.045 normals PK4 Healthy 0.025 0.069 0.095 0.139 0.018 0.028 0.036 0.040 normals
Table 5.3b Optimization of rTES-32 recombinant antigen concentration and serum dilution in IgG4-ELISA
Antigen conc. (μg/ml) 5 10 20 40 5 10 20 40 Serum Serum Code dilution 1:50 1:50 1:50 1:50 1:100 1:100 1:100 1:100 Serum description T2 Toxocariasis 0.18 0.20 0.23 0.24 0.098 0.152 0.188 0.216 2 0 2 2 T5 Toxocariasis 1.07 1.33 1.87 1.80 0.532 0.712 0.830 0.925 2 4 4 7 T7 Toxocariasis 1.12 1.29 1.33 1.23 0.615 0.721 0.825 0.900 9 9 6 9 T12 Toxocariasis 0.21 0.30 0.33 0.39 0.190 0.218 0.315 0.325 0 0 9 9 PK1 Healthy 0.02 0.03 0.05 0.08 0.011 0.017 0.033 0.049 normals 1 0 4 5 PK2 Healthy 0.06 0.07 0.80 0.12 0.039 0.055 0.025 0.055 normals 6 4 8 PK3 Healthy 0.02 0.03 0.08 0.08 0.009 0.015 0.018 0.066 normals 0 2 5 5 PK4 Healthy 0.03 0.03 0.08 0.08 0.013 0.022 0.034 0.078 normals 0 9 2 9
Figures in bold show the optimum concentrations of rTES-26 and rTES-32 recombinant antigens and serum dilutions.
217 Table 5.3c Optimization of rTES-120 recombinant antigen concentration and serum dilution in IgG4-ELISA
Antigen conc. (μg/ml) 5 10 20 40 5 10 20 40 Serum Serum Code dilution 1:50 1:50 1:50 1:50 1:100 1:100 1:100 1:100 Serum description T2 Toxocariasis 0.512 0.610 0.699 0.800 0.244 0.418 0.450 0.510 T5 Toxocariasis 1.783 1.878 1.871 1.117 1.182 1.253 0.760 0.808 T7 Toxocariasis 1.102 1.451 1.512 1.421 0.793 0.778 0.470 0.475 T12 Toxocariasis 1.928 2.706 2.715 2.800 1.112 1.522 1.539 1.712 PK1 Healthy 0.085 0.125 0.175 0.215 0.100 0.102 0.079 0.090 normals PK2 Healthy 0.038 0.067 0.100 0.128 0.066 0.067 0.063 0.069 normals PK3 Healthy 0.032 0.068 0.124 0.175 0.009 0.012 0.058 0.100 normals PK4 Healthy 0.044 0.085 0.164 0.199 0.033 0.041 0.102 0.156 normals
Figure in bold shows the optimum concentrations of rTES-120 recombinant antigen and serum dilutions.
218 Table 5.3d Optimization of the dilutions of anti-human IgG4 antibody conjugated to HRP in IgG4-ELISA
rTES-26 rTES-32 rTES-120 Serum 1:500 1:1000 1:2000 1:500 1:1000 1:2000 1:500 1:1000 1:2000 Code T2 0.812 0.717 0.312 0.418 0.232 0.128 0.925 0.610 0.299 T5 2.355 2.077 1.199 2.218 1.874 0.918 1.936 1.878 0.949 T7 2.121 1.993 0.835 1.992 1.336 0.695 1.825 1.457 0.897 T12 3.500 3.490 2.125 0.582 0.339 0.199 2.895 2.706 1.925 PK1 0.095 0.051 0.045 0.121 0.054 0.022 0.231 0.125 0.089 PK2 0.151 0.095 0.082 0.209 0.128 0.009 0.072 0.067 0.054 PK3 0.108 0.050 0.048 0.158 0.085 0.069 0.123 0.068 0.042 PK4 0.129 0.069 0.060 0.149 0.082 0.060 0.154 0.085 0.051
Figures in bold show the optimum dilution of anti-human IgG4 antibody for each recombinant antigen.
Note: T = Toxocariasis, PK = Healthy normals
219 Table 5.4 Optimization of antigen concentrations and conjugate dilutions in IgG1-ELISA
(A) rTES-26 Antigen conc. 5 µg/ml 10 µg/ml 20 µg/ml (μg/ml) Conjugate 1:10,000 1:15,000 1:10,000 1:15,000 1:10,000 1:15,000 dilution Serum code
PK 2 0.218 0.099 0.493 0.327 0.348 0.295 PK 3 0.231 0.129 0.364 0.263 0.398 0.282 PK 4 0.294 0.178 0.507 0.281 0.531 0.321 PK 7 0.546 0.299 0.939 0.558 1.049 0.701 PK 12 0.234 0.102 0.266 0.195 0.297 0.199 T2 0.744 0.425 0.954 0.617 0.937 0.612 T4 0.967 0.612 1.242 0.813 1.208 0.804 T5 1.711 1.278 2.596 1.332 2.717 1.355 T7 1.788 1.299 2.428 1.228 2.389 1.240 T9 0.859 0.597 1.126 0.625 1.098 0.699
(B) rTES-32 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:10,000 1:15,000 1:10,000 1:15,000 1:10,000 1:15,000 PK 2 0.318 0.129 0.412 0.226 0.425 0.236 PK 3 0.221 0.121 0.396 0.216 0.402 0.222 PK 4 0.214 0.098 0.375 0.199 0.399 0.200 PK 7 0.246 0.169 0.385 0.287 0.395 0.299 PK 12 0.134 0.082 0.234 0.136 0.310 0.189 T2 0.323 0.298 0.488 0.309 0.558 0.345 T4 0.966 0.569 1.009 0.625 1.025 0.710 T5 1.855 1.299 2.015 1.468 2.119 1.425 T7 0.964 0.598 1.045 0.612 1.125 0.700 T9 0.324 0.253 0.381 0.266 0.399 0.251
(C) rTES-120 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:10,000 1:15,000 1:10,000 1:15,000 1:10,000 1:15,000 PK 2 0.121 0.082 0.156 0.110 0.169 0.186 PK 3 0.214 0.194 0.234 0.228 0.245 0.234 PK 4 0.246 0.208 0.284 0.221 0.297 0.223 PK 7 0.034 0.012 0.059 0.021 0.069 0.035 PK 12 0.244 0.127 0.298 0.199 0.312 0.208 T2 0.668 0.522 0.701 0.532 0.699 0.499 T4 0.780 0.688 0.798 0.699 0.745 0.658 T5 1.412 1.245 1.456 1.234 1.392 1.118 T7 1.625 1.421 1.697 1.477 1.645 1.399 T9 0.789 0.618 0.800 0.611 0.810 0.625
Note: T = Toxocariasis, PK = Healthy normals IgG1-HRP (serum dilution 1:50) Figures in bold show the optimum concentrations of recombinant antigens and conjugate dilutions.
220 Table 5.5 Optimization of antigen concentrations and conjugate dilutions in IgG2-ELISA
(A) rTES-26 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.222 0.125 0.231 0.139 0.318 0.199 PK 3 0.090 0.041 0.125 0.065 0.231 0.112 PK 4 0.058 0.030 0.070 0.045 0.143 0.082 PK 7 0.032 0.011 0.032 0.025 0.074 0.054 PK 12 0.138 0.095 0.218 0.115 0.299 0.180 T2 0.239 0.197 0.345 0.225 0.340 0.230 T4 0.377 0.202 0.390 0.248 0.400 0.310 T5 1.218 0.928 1.675 1.452 1.669 1.469 T7 1.421 1.125 1.700 1.512 1.699 1.528 T9 0.382 0.245 0.410 0.330 0.488 0.340
(B) rTES-32 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.165 0.092 0.178 0.095 0.273 0.148 PK 3 0.082 0.053 0.092 0.042 0.124 0.067 PK 4 0.041 0.015 0.040 0.012 0.092 0.048 PK 7 0.228 0.110 0.230 0.115 0.398 0.220 PK 12 0.049 0.018 0.058 0.025 0.120 0.058 T2 0.125 0.085 0.228 0.122 0.357 0.208 T4 0.340 0.291 0.514 0.351 0.680 0.412 T5 1.622 1.312 1.810 1.628 2.012 1.630 T7 1.420 1.109 1.500 1.322 1.855 1.299 T9 0.592 0.425 0.605 0.588 0.620 0.590
(C) rTES-120 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.040 0.022 0.045 0.024 0.078 0.040 PK 3 0.205 0.125 0.286 0.129 0.385 0.210 PK 4 0.211 0.100 0.224 0.112 0.289 0.185 PK 7 0.168 0.089 0.175 0.099 0.234 0.143 PK 12 0.097 0.030 0.100 0.035 0.138 0.072 T2 0.499 0.286 0.567 0.327 0.622 0.312 T4 1.804 1.512 1.824 1.611 1.812 1.620 T5 0.732 0.697 0.799 0.781 0.826 0.788 T7 1.102 0.924 1.299 1.115 1.300 1.015 T9 0.613 0.345 0.705 0.471 0.716 0.481
Note: T = Toxocariasis, PK = Healthy normals IgG2-HRP (serum dilution 1:50)
Figures in bold show the optimum concentrations of recombinant antigens and conjugate dilutions.
221 Table 5.6 Optimization of antigen concentrations and conjugate dilutions in IgG3-ELISA
(A) rTES-26 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.357 0.180 0.419 0.299 0.499 0.363 PK 3 1.340 0.895 1.639 0.925 1.219 0.754 PK 4 0.396 0.284 0.483 0.298 0.393 0.271 PK 7 0.415 0.322 0.484 0.345 0.310 0.211 PK 12 0.734 0.428 0.771 0.445 0.721 0.423 T2 1.966 1.125 2.095 1.242 2.078 1.285 T4 0.092 0.042 0.127 0.085 0.096 0.067 T5 0.071 0.058 0.100 0.055 0.060 0.048 T7 0.790 0.434 0.808 0.487 0.769 0.425 T9 2.394 1.812 2.435 1.887 2.453 1.899
(B) rTES-32 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.020 0.029 0.035 0.015 0.041 0.038 PK 3 0.102 0.051 0.123 0.089 0.131 0.089 PK 4 0.087 0.038 0.096 0.045 0.106 0.055 PK 7 0.045 0.035 0.056 0.024 0.088 0.046 PK 12 0.056 0.044 0.089 0.064 0.104 0.078 T2 0.125 0.082 0.131 0.099 0.139 0.100 T4 0.088 0.045 0.100 0.058 0.125 0.055 T5 0.875 0.645 1.006 0.896 0.997 0.724 T7 0.880 0.698 1.125 0.995 1.007 0.984 T9 1.005 0.921 1.545 1.355 1.550 1.299
(C) rTES-120 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.110 0.070 0.153 0.092 0.190 0.115 PK 3 0.182 0.099 0.210 0.112 0.241 0.178 PK 4 0.192 0.100 0.232 0.125 0.272 0.212 PK 7 0.123 0.082 0.105 0.054 0.218 0.104 PK 12 0.208 0.158 0.240 0.134 0.298 0.165 T2 0.094 0.058 0.120 0.065 0.122 0.082 T4 0.542 0.310 0.624 0.425 0.645 0.433 T5 1.003 0.628 1.599 1.345 1.925 1.542 T7 1.243 0.911 1.623 1.423 2.015 1.620 T9 0.425 0.240 0.722 0.564 0.842 0.571
Note: T = Toxocariasis, PK = Healthy normals IgG3-HRP (serum dilution 1:50)
Figures in bold show the optimum concentrations of recombinant antigens and conjugate dilutions.
222 Table 5.7 Optimization of antigen concentrations and conjugate dilutions in IgE-ELISA
(A) rTES-26 Serum Code 2.5 µg/ml 5 µg/ml 10 µg/ml 1:1000 1:2000 1:1000 1:2000 1:1000 1:2000 PK 2 0.185 0.099 0.239 0.134 0.245 0.155 PK 3 0. 510 0.260 0.618 0.482 0.622 0.489 PK 4 0.480 0.265 0.599 0.328 0.610 0.345 PK 7 0.175 0.055 0.258 0.115 0.259 0.128 PK 12 0.362 0.168 0.485 0.248 0.498 0.050 T2 0.619 0.412 0.622 0.454 0.738 0.623 T4 0.584 0.246 0.481 0.255 0.499 0.261 T5 0.497 0.317 0.697 0.471 0.691 0.467 T7 0.475 0.253 0.599 0.365 0.620 0.399
(B) rTES-32 Serum Code 2.5 µg/ml 5 µg/ml 10 µg/ml 1:1000 1:2000 1:1000 1:2000 1:1000 1:2000 PK 2 0.123 0.089 0.135 0.096 0.148 0.104 PK 3 0.382 0.245 0.399 0.264 0.415 0.268 PK 4 0.080 0.065 0.098 0.072 0.107 0.086 PK 7 0.086 0.076 0.100 0.087 0.105 0.090 PK 12 0.086 0.066 0.099 0.078 0.112 0.089 T2 0.388 0.256 0.398 0.266 0.399 0.270 T4 0.210 0.165 0.220 0.178 0.228 0.185 T5 0.399 0.243 0.405 0.255 0.400 0.250 T7 0.524 0.376 0.550 0.370 0.545 0.368 T9 0.029 0.018 0.035 0.022 0.034 0.021
(C) rTES-120 Serum Code 2.5 µg/ml 5 µg/ml 10 µg/ml 1:1000 1:2000 1:1000 1:2000 1:1000 1:2000 PK 2 0.239 0.199 0.257 0.214 0.261 0.199 PK 3 0.282 0.203 0.296 0.212 0.320 0.215 PK 4 0.280 0.210 0.299 0.235 0.317 0.244 PK 7 0.068 0.040 0.107 0.057 0.130 0.099 PK 12 0.108 0.052 0.154 0.095 0.163 0.122 T2 0.425 0.245 0.456 0.266 0.466 0.274 T4 0.392 0.220 0.399 0.229 0.395 0.236 T5 0.400 0.316 0.413 0.320 0.420 0.315 T7 0.699 0.426 0.710 0.432 0.705 0.440 T9 0.048 0.027 0.077 0.045 0.092 0.051
Note: T = Toxocariasis, PK = Healthy normals IgE-HRP (serum dilution 1:50)
Figures in bold show the optimum concentrations of recombinant antigens and conjugate dilutions.
223 Table 5.8 Optimization of antigen concentrations and conjugate dilutions in IgM-ELISA
(A) rTES-26 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.120 0.085 0.158 0.100 0.155 0.106 PK 5 0.399 0.300 0.424 0.326 0.430 0.320 PK 9 0.200 0.187 0.255 0.198 0.269 0.200 PK 44 0.425 0.310 0.466 0.346 0.478 0.357 T2 0.020 0.024 0.035 0.033 0.040 0.036 T4 0.024 0.017 0.055 0.043 0.056 0.048 T5 0.047 0.030 0.092 0.053 0.100 0.059 T7 0.100 0.099 0.155 0.128 0.160 0.125
(B) rTES-32 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.099 0.080 0.125 0.094 0.169 0.130 PK 5 0.499 0.308 0.670 0.407 0.723 0.500 PK 9 0.199 0.150 0.210 0.156 0.355 0.260 PK 44 0.600 0.450 0.699 0.456 0.789 0.588 T2 0.085 0.030 0.100 0.039 0.150 0.055 T4 0.150 0.050 0.180 0.081 0.200 0.099 T5 0.080 0.070 0.099 0.096 0.105 0.100 T12 0.550 0.435 0.899 0.677 0.890 0.699
(C) rTES-120 Serum Code 5 µg/ml 10 µg/ml 20 µg/ml 1:500 1:1000 1:500 1:1000 1:500 1:1000 PK 2 0.200 0.155 0.255 0.174 0.266 0.199 PK 5 0.245 0.199 0.310 0.245 0.320 0.250 PK 9 0.760 0.423 0.824 0.503 0.833 0.511 PK 44 0.680 0.312 0.766 0.425 0.787 0.430 T2 0.200 0.100 0.230 0.146 0.231 0.150 T4 0.320 0.199 0.342 0.277 0.330 0.270 T5 0.416 0.310 0.530 0.402 0.535 0.400 T7 0.180 0.088 0.200 0.123 0.211 0.125
Note: T = Toxocariasis, PK = Healthy normals IgM-HRP (serum dilution 1:50)
Figures in bold show the optimum concentrations of recombinant antigens and conjugate dilutions.
224 Table 5.9 Optimum conditions for each rTES ELISA assay
Optimized Antigen Serum Conjugate parameters concentration dilution dilution s (µg/ml) factor factor
rTES-26 ELISA IgG1-HRP 5 1:15,000 IgG2-HRP 10 1:1000 IgG3-HRP 10 1:50 1:1000 IgG4-HRP 10 1:1000 IgE-HRP 5 1:2000 IgM-HRP 10 1:1000 rTES-32 ELISA IgG1-HRP 5 1:15,000 IgG2-HRP 10 1:1000 IgG3-HRP 10 1:50 1:1000 IgG4-HRP 20 1:1000 IgE-HRP 2.5 1:1000 IgM-HRP 10 1:1000 rTES-120 ELISA IgG1-HRP 5 1:15,000 IgG2-HRP 10 1:1000 IgG3-HRP 10 1:50 1:1000 IgG4-HRP 10 1:1000 IgE-HRP 2.5 1:1000 IgM-HRP 10 1:1000
225 5.4.3 Sensitivity and specificity analysis of recombinant antigens using IgG1, IgG2,
IgG3, IgE and IgM
The sensitivity and specificity of each assay are shown in Tables 5.10a – 5.10c. The summary of evaluations of the sensitivities and specificities are shown in Table 5.11.
Substantial cross-reactivities were demonstrated for all IgG subclasses (IgG1, IgG2 and
IgG3) and IgE with cross-reactions shown by serum samples in all non-toxocara groups.
IgG1 gave the greatest cross-reactivities (33.8-36.9%) thus least specific, followed by IgG2
(24.6-33.8%), IgG3 (21.5-27.7%), IgE (23-26.2%) and IgM (16.7-33.3%). Eventhough IgM assay gave less cross-reactivities, the sensitivity of this assay was very low. Out of 12
Toxocara patients tested, only one Toxocara sample was found to be positive by rTES-26
(8.3%) and rTES-32 (8.3%) and four positive by rTES-120 (33.3%) by the IgM assay.
The assay using rTES-26 gave the least cross-reactivity, hence the highest specificity as compared to rTES-32 and rTES-120 in all assays. With regards to sensitivity, rTES-120 gave the highest value in all assays. Based on the sensitivity and specificity results, IgG4 was chosen as the best secondary antibody to be used in the development of an immunoassay for detection of Toxocara infection.
226 Table 5.10a Sensitivity and specificity analysis of rTES-26 ELISA
No Serum Code IgG1 IgG2 IgG3 IgE IgM 1. T1 0.120 0.078 0.098 0.022 0.056 2. T2 0.425 0.225 0.966 0.454 0.033 3. T3 0.100 0.009 0.068 0.158 ND 4. T4 0.612 0.248 0.392 0.255 0.043 5. T5 1.278 1.452 1.058 0.471 0.053 6. T6 0.099 0.340 0.067 0.009 0.015 7. T7 1.299 1.512 0.790 0.365 0.122 8. T8 0.002 0.173 0.005 0.010 0.128 9. T9 0.597 0.330 1.394 0.043 0.058 10. T10 0.099 0.100 0.087 0.020 0.112 11. T12 3.490 0.246 1.250 1.242 0.565 12. T15 0.425 0.329 0.229 0.825 ND 13. UMT#3 0.065 0.156 0.056 0.743 ND 14. UMT#6 0.123 0.092 0.097 0.098 ND 15. UMT#9 0.234 0.249 0.269 0.129 ND 16. MBDR60 0.165 0.371 0.128 0.542 ND 17. MBRD62 0.068 0.054 0.167 0.042 ND 18. MBDR66 0.098 0.129 0.122 0.071 ND 19. T16 0.133 0.132 0.019 0.423 0.091 20. T17 0.125 0.341 0.154 0.348 0.100 21. PK 1 0.132 0.165 0.106 0.028 ND 22. PK 2 0.099 0.139 0.015 0.134 0.100 23. PK 3 0.129 0.065 0.035 0.482 ND 24. PK 4 0.178 0.045 0.064 0.328 ND 25. PK 5 0.109 0.389 0.278 0.032 0.326 26. PK 6 0.088 0.178 0.050 0.083 ND 27. PK 7 0.299 0.025 0.020 0.115 ND 28. PK 8 0.125 0.299 0.215 0.123 ND 29. PK 9 0.178 0.134 0.010 0.092 0.190 30. PK 10 0.081 0.085 0.011 0.043 ND 31. PK 11 0.025 0.025 0.126 0.248 ND 32. PK 12 0.102 0.115 0.099 0.081 ND 33. PK 13 0.047 0.037 0.064 0.145 ND 34. PK 14 0.088 0.091 0.035 0.138 ND 35. PK 15 0.055 0.068 0.033 0.022 ND 36. PK 16 0.123 0.068 0.098 0.041 ND 37. PK 17 0.092 0.099 0.066 0.039 ND 38. PK 30 0.178 0.124 0.129 0.091 ND 39. PK 36 0.081 0.008 0.058 0.023 ND 40. PK 44 0.219 0.465 0.348 0.523 0.346 41. Tri 1 0.129 0.105 0.164 0.878 ND 42. Tri 5 1.158 0.890 1.005 0.622 0.034 43. Tri 7 0.664 0.253 0.026 0.925 0.018 44. Tri 10 0.133 0.120 0.128 0.139 0.146
227 Table 5.10a (cont.) Sensitivity and specificity analysis of rTES-26 ELISA
45. Tri 12 0.394 0.455 0.668 1.082 0.091 46. Asc 1 0.045 0.099 0.098 0.039 0.118 47. Asc 2 0.055 0.077 0.060 0.053 0.023 48. Asc 3 0.369 0.374 0.041 0.129 ND 49. Asc 7 0.089 0.084 0.020 0.125 0.221 50. Asc 9 0.122 0.074 0.125 0.152 ND 51. Asc 10 0.266 0.097 0.299 0.018 0.011 52. Mix 17 0.361 0.125 0.056 0.023 0.043 53. Mix 18 0.269 0.089 0.123 0.017 0.873 54. Mix 19 0.300 0.097 0.025 0.112 0.047 55. Nec 2 0.176 0.325 0.340 0.081 ND 56. EH74 0.157 0.143 0.180 0.738 0.107 57. EH70 0.180 0.185 0.168 0.026 0.015 58. EH66 0.312 0.152 0.336 0.243 0.024 59. EH29 0.100 0.435 0.035 0.033 0.070 60. EH11 0.303 0.054 0.036 0.998 0.051 61. EH77 0.114 0.224 0.011 0.052 ND 62. EH79 0.283 0.358 0.425 0.025 ND 63. EH53 0.134 0.080 0.124 0.093 ND 64. EH56 0.600 0.122 0.100 0.052 ND 65. EH42 0.078 0.092 0.020 0.012 ND 66. UMT70 0.243 0.566 0.425 0.212 ND 67. UMT83 0.397 0.164 0.155 0.018 ND 68. UMT45 0.068 0.099 0.068 0.075 ND 69. UMT66 0.099 0.080 0.121 0.032 ND 70. UMT90 0.087 0.055 0.031 0.012 ND 71. UMT6 0.421 0.488 0.522 0.427 0.128 72. UMT30 0.121 0.057 0.124 0.058 ND 73. UMT21 0.282 0.351 0.397 0.108 0.289 74. UMT77 0.285 0.122 0.123 0.091 0.096 75. UMT49 0.386 0.102 0.100 0.035 0.031 76. SB57 0.096 0.034 0.099 0.289 ND 77. SB31 0.172 0.089 0.003 0.282 0.141 78. SB9 0.341 0.485 0.425 0.322 0.116 79. SB58 0.294 0.099 0.002 0.018 0.074 80. SB21 0.131 0.067 0.003 0.124 0.065 81. SB17 0.077 0.088 0.051 0.099 ND 82. SB46 0.163 0.126 0.131 0.015 ND 83. SB13 0.119 0.124 0.120 0.068 ND 84. SB52 0.315 0.544 0.396 0.145 0.099 85. SBSin83 0.126 0.111 0.009 0.013 ND
Note: No. 1 – 20:Toxocariasis; 21 – 40:Healthy normals; 41 – 55:STH; 56 – 65:Amoebiasis; 66 –75:Toxoplasmosis; 76 – 85: B. malayi microfilaremic
Numbers in bold represent positive; bold and highlighted represent false positive cases;ND : not done
228 Table 5.10b Sensitivity and specificity analysis of rTES-32 ELISA
No Serum Code IgG1 IgG2 IgG3 IgE IgM 1 T1 0.099 0.128 0.122 0.025 0.034 2 T2 0.298 0.122 0.099 0.388 0.039 3 T3 0.096 0.017 0.023 0.140 ND 4 T4 0.569 0.351 0.058 0.210 0.081 5 T5 1.299 1.628 0.896 0.399 0.096 6 T6 0.062 0.096 0.035 0.012 0.045 7 T7 0.598 1.322 0.995 0.524 0.145 8 T8 0.002 0.147 0.124 0.018 0.182 9 T9 0.253 0.588 0.236 0.029 0.047 10 T10 0.003 0.181 0.100 0.058 0.100 11 T12 1.225 0.462 1.355 0.768 0.667 12 T15 0.525 1.192 0.678 0.622 ND 13 UMT#3 0.032 0.369 0.059 0.328 ND 14 UMT#6 0.005 0.477 0.158 0.042 ND 15 UMT#9 0.123 0.359 0.323 0.138 ND 16 MBDR60 0.451 0.350 0.453 0.389 ND 17 MBRD62 0.111 0.411 0.252 0.082 ND 18 MBDR65 0.085 0.055 0.098 0.099 ND 19 MBDR66 0.123 0.126 0.145 0.392 0.120 20 T17 0.065 0.019 0.065 0.421 0.033 21 PK 1 0.038 0.088 0.036 0.088 ND 22 PK 2 0.129 0.095 0.015 0.123 0.094 23 PK 3 0.121 0.042 0.089 0.382 ND 24 PK 4 0.098 0.012 0.045 0.080 ND 25 PK 5 0.025 0.309 0.007 0.031 0.407 26 PK 6 0.066 0.045 0.016 0.018 ND 27 PK 7 0.169 0.015 0.024 0.086 ND 28 PK 8 0.088 0.478 0.289 0.164 ND 29 PK 9 0.035 0.069 0.066 0.134 0.156 30 PK 10 0.125 0.125 0.167 0.071 ND 31 PK 11 0.163 0.148 0.110 0.086 ND 32 PK 12 0.082 0.125 0.064 0.126 ND 33 PK 13 0.045 0.089 0.034 0.079 ND 34 PK 14 0.029 0.045 0.035 0.543 ND 35 PK 15 0.036 0.068 0.012 0.044 ND 36 PK 16 0.058 0.064 0.025 0.091 ND 37 PK 17 0.099 0.034 0.036 0.089 ND 38 PK 30 0.115 0.066 0.087 0.078 ND 39 PK 36 0.092 0.081 0.126 0.082 ND 40 PK 44 0.225 0.386 0.355 0.092 0.456 41 Tri 1 0.136 0.186 0.156 0.925 ND 42 Tri 5 0.998 1.616 1.253 0.799 0.064 43 Tri 7 0.335 0.068 0.056 0.051 0.028 44 Tri 10 0.066 0.098 0.125 1.825 0.066
229 Table 5.10b (cont.) Sensitivity and specificity analysis of rTES-32 ELISA
45 Tri 12 0.285 0.013 0.099 0.011 0.096 46 Asc 1 0.097 0.121 0.129 0.048 0.106 47 Asc 2 0.064 0.179 0.145 0.125 0.075 48 Asc 3 0.569 0.285 0.158 0.043 ND 49 Asc 7 0.653 0.319 0.465 0.873 0.245 50 Asc 9 0.189 0.286 0.299 0.128 ND 51 Asc 10 0.299 0.386 0.059 0.053 0.012 52 Mix 17 0.552 0.528 0.822 0.248 0.033 53 Mix 18 0.265 0.316 0.423 0.125 0.756 54 Mix 19 0.155 0.106 0.097 0.095 0.040 55 Nec 2 0.152 0.528 0.154 0.048 ND 56 EH74 0.121 0.059 0.085 1.435 0.089 57 EH70 0.125 0.074 0.021 0.129 0.034 58 EH66 0.355 0.474 0.356 0.513 0.012 59 EH29 0.026 1.728 1.223 0.092 0.027 60 EH11 0.312 0.458 0.219 0.049 0.055 61 EH77 0.099 0.827 0.087 0.145 ND 62 EH79 0.286 1.226 0.456 0.153 ND 63 EH53 0.085 0.047 0.022 0.180 ND 64 EH56 0.552 0.166 0.009 0.058 ND 65 EH42 0.034 0.105 0.010 0.092 ND 66 UMT70 0.031 0.182 0.031 0.315 ND 67 UMT83 0.084 0.330 0.042 0.123 ND 68 UMT45 0.145 0.158 0.050 0.052 ND 69 UMT66 0.138 0.141 0.021 0.048 ND 70 UMT90 0.544 0.854 0.569 0.032 ND 71 UMT6 0.390 0.226 0.223 0.588 0.108 72 UMT30 0.032 0.036 0.086 0.018 ND 73 UMT21 0.009 0.056 0.002 0.128 0.380 74 UMT88 0.009 0.091 0.007 0.167 0.087 75 UMT49 0.025 0.076 0.031 0.092 0.055 76 SB57 0.035 0.023 0.011 0.329 ND 77 SB31 0.058 0.183 0.007 0.410 0.145 78 SB9 0.086 0.123 0.099 0.390 0.100 79 SB58 0.098 0.138 0.113 0.019 0.094 80 SB21 0.167 0.170 0.146 0.080 0.035 81 SB17 0.064 0.029 0.012 0.033 ND 82 SB46 0.042 0.021 0.022 0.059 ND 83 SB13 0.335 0.314 0.488 0.018 0.299 84 SB52 0.155 0.170 0.137 0.124 ND 85 SBSin83 0.031 0.040 0.020 0.185 ND
Note: No. 1 – 20:Toxocariasis; 21 – 40:Healthy normals; 41 – 55:STH; 56 – 65:Amoebiasis; 66 –75:Toxoplasmosis; 76 – 85: B. malayi microfilaremic
Numbers in bold represents positive; bold and highlighted represent false positive cases.;ND: not done
230 Table 5.10c Sensitivity and specificity analysis of rTES-120 ELISA
No Serum Code IgG1 IgG2 IgG3 IgE IgM 1 T1 0.120 0.036 0.098 0.099 0.356 2 T2 0.425 0.327 0.065 0.425 0.146 3 T3 0.100 0.133 0.122 0.189 ND 4 T4 0.612 1.611 0.425 0.392 0.277 5 T5 1.278 0.781 1.345 0.400 0.402 6 T6 0.925 1.825 0.899 0.091 0.123 7 T7 1.200 1.115 1.423 0.699 0.156 8 T8 0.052 0.164 0.023 0.055 0.190 9 T9 0.582 0.471 0.055 0.048 0.087 10 T10 0.099 0.118 0.091 0.129 0.390 11 T12 3.490 0.368 0.564 0.789 0.766 12 T15 0.120 0.092 0.185 0.599 ND 13 UMT#3 0.425 0.354 0.234 0.330 ND 14 UMT#6 0.100 0.237 0.122 0.023 ND 15 UMT#9 0.612 0.297 0.339 0.121 ND 16 MBDR60 1.278 0.299 0.285 0.391 ND 17 MBRD62 0.099 0.518 0.410 0.023 ND 18 MBDR65 0.120 0.302 0.097 0.077 ND 19 MBDR66 0.025 0.378 0.120 0.399 0.012 20 T17 0.031 0.425 0.064 0.768 0.377 21 PK 1 0.055 0.081 0.077 0.134 ND 22 PK 2 0.082 0.024 0.092 0.239 0.174 23 PK 3 0.164 0.129 0.112 0.282 ND 24 PK 4 0.208 0.112 0.125 0.280 ND 25 PK 5 0.058 0.121 0.082 0.062 0.245 26 PK 6 0.068 0.100 0.071 0.038 ND 27 PK 7 0.012 0.099 0.054 0.068 ND 28 PK 8 0.120 0.154 0.322 0.146 ND 29 PK 9 0.036 0.007 0.010 0.184 0.503 30 PK 10 0.099 0.084 0.038 0.017 ND 31 PK 11 0.125 0.056 0.083 0.071 ND 32 PK 12 0.127 0.035 0.134 0.108 ND 33 PK 13 0.058 0.065 0.093 0.097 ND 34 PK 14 0.082 0.123 0.081 0.122 ND 35 PK 15 0.152 0.287 0.074 0.012 ND 36 PK 16 0.036 0.069 0.012 0.020 ND 37 PK 17 0.047 0.054 0.028 0.040 ND 38 PK 30 0.192 0.299 0.355 0.058 ND 39 PK 36 0.115 0.112 0.123 0.018 ND 40 PK 44 0.312 0.455 0.510 0.458 0.425 41 Tri 1 0.142 0.102 0.054 1.345 ND 42 Tri 5 0.835 0.406 0.582 0.813 0.194 43 Tri 7 0.473 0.403 0.801 1.008 0.160 44 Tri 10 0.010 0.156 0.020 1.245 0.463 45 Tri 11 0.339 0.132 0.125 1.230 0.065 46 Asc 1 0.035 0.020 0.022 0.145 0.462 47 Asc 2 0.056 0.041 0.010 0.034 0.104
231 Table 5.10c (cont.) Sensitivity and specificity analysis of rTES-120 ELISA
48 Asc 3 0.500 0.454 0.625 0.825 ND 49 Asc 7 0.229 0.448 0.092 0.020 0.534 50 Asc 9 0.152 0.386 0.110 0.068 ND 51 Asc 10 0.325 0.414 0.345 0.025 0.121 52 Mix 17 0.518 0.603 0.628 0.099 0.129 53 Mix 18 0.423 0.022 0.035 0.085 0.765 54 Mix 19 0.117 0.104 0.012 0.015 0.140 55 Nec 2 0.122 0.424 0.044 0.050 ND 56 EH74 0.162 0.024 0.009 1.242 0.264 57 EH70 0.178 0.029 0.100 0.083 0.140 58 EH66 0.395 0.237 0.381 0.918 0.173 59 EH29 0.225 0.213 0.030 0.143 0.133 60 EH11 0.428 0.897 0.528 0.822 0.151 61 EH77 0.125 0.358 0.412 0.048 ND 62 EH79 0.393 0.224 0.388 0.012 ND 63 EH53 0.100 0.037 0.012 0.008 ND 64 EH56 0.825 0.008 0.009 0.010 ND 65 EH42 0.021 0.159 0.041 0.095 ND 66 UMT70 0.423 0.112 0.122 0.455 ND 67 UMT83 0.285 0.349 0.142 0.020 ND 68 UMT45 0.013 0.171 0.083 0.122 ND 69 UMT66 0.006 0.133 0.014 0.099 ND 70 UMT90 0.015 0.513 0.322 0.040 ND 71 UMT6 0.514 0.481 0.288 1.982 0.018 72 UMT30 0.006 0.021 0.018 0.092 ND 73 UMT21 0.287 0.152 0.348 0.123 0.034 74 UMT88 0.024 0.025 0.032 0.182 0.064 75 UMT49 0.138 0.002 0.125 0.123 0.012 76 SB57 0.035 0.024 0.018 0.925 ND 77 SB31 0.185 0.014 0.031 0.434 0.268 78 SB9 0.328 0.177 0.375 0.568 0.134 79 SB58 0.282 0.047 0.042 0.081 0.238 80 SB21 0.014 0.037 0.009 0.125 0.183 81 SB17 0.012 0.021 0.011 0.238 ND 82 SB46 0.162 0.067 0.018 0.027 ND 83 SB13 0.132 0.225 0.310 0.012 ND 84 SB52 0.148 0.058 0.007 1.122 0.212 85 SBSin83 0.009 0.037 0.020 0.044 ND
Note: No. 1 – 20: Toxocariasis; 21 – 40: Healthy normals; 41 – 55: STH; 56 – 65:Amoebiasis; 66 –75:Toxoplasmosis; 76 – 85: B. malayi microfilaremic
Numbers in bold represent positive cases. Numbers in bold and highlighted represent false positive cases. ND: not done
232 Table 5.11 Sensitivity and cross-reaction of sera from patients with other parasitic infections in rTES ELISA using IgG subclasses, IgE and IgM
(a) IgG1-ELISA Type of serum rTES-26 rTES-32 rTES-120 Sensitivity Toxocariasis 8/20 (40%) 8/20 (40%) 10/20 (50%) Cross-reactivities STH 8/15 8/15 8/15 B. malayi microfilaremics 3/10 3/10 3/10 Amoebiasis 4/10 5/10 5/10 Toxoplasmosis 6/10 6/10 6/10 Healthy normals 1/20 1/20 2/20 Total % of cross reactivity 22/65 (33.8%) 23/65 (35.4%) 24/65 (36.9%)
(b) IgG2-ELISA Type of serum rTES-26 rTES-32 rTES-120 Sensitivity Toxocariasis 11/20 (55%) 11/20 (55%) 15/20 (75%) Cross-reactivities STH 5/15 8/15 8/15 B. malayi microfilaremics 2/10 2/10 2/10 Amoebiasis 3/10 5/10 5/10 Toxoplasmosis 3/10 3/10 4/10 Healthy normals 3/20 3/20 3/20 Total % of cross reactivity 16/65 (24.6%) 21/65 (32.3%) 22/65 (33.8%)
(c) IgG3-ELISA Type of serum rTES-26 rTES-32 rTES-120 Sensitivity Toxocariasis 8/20 (40%) 8/20 (40%) 9/20 (45%) Cross-reactivities STH 4/15 5/15 5/15 B. malayi microfilaremics 2/10 2/10 2/10 Amoebiasis 2/10 4/10 4/10 Toxoplasmosis 3/10 5/10 4/10 Healthy normals 3/20 2/20 3/20 Total % of cross reactivity 14/65 (21.5%) 18/65 (27.7%) 18/65 (27.7%)
233 Table 5.11(cont.) Sensitivity and cross-reaction of sera from patients with other parasitic infections in rTES-ELISA using IgG subclasses, IgE and IgM
(d) IgE-ELISA Type of serum rTES-26 rTES-32 rTES-120 Sensitivity Toxocariasis 10/20 (50%) 11/20 (55%) 11/20 (55%) Cross-reactivities STH 4/15 5/15 5/15 B. malayi microfilaremics 3/10 3/10 3/10 Amoebiasis 3/10 3/10 3/10 Toxoplasmosis 2/10 3/10 3/10 Healthy normals 3/20 3/20 3/20 Total % of cross reactivity 15/65 (23%) 17/65 (26.2%) 17/65 (26.2%)
(e) IgM-ELISA Type of serum rTES-26 rTES-32 rTES-120 Sensitivity Toxocariasis 1/12 (8.33%) 1/12 (8.33%) 4/12 (33.3%) Cross-reactivities STH 2/11 2/11 3/11 B. malayi microfilaremics 0/5 1/5 3/5 Amoebiasis 0/5 0/5 1/5 Toxoplasmosis 1/4 1/4 0/4 Healthy normals 2/5 2/5 3/5 Total % of cross reactivity 5/30 (16.7%) 6/30 (20%) 10/30 (33.3%)
234 5.3.4 Sensitivity and specificity analysis of recombinant antigens using IgG4-ELISA
The sensitivity using rTES-26, rTES-32, rTES-120 and rTES-30USM are shown in Table
5.12. The overall evaluations of the sensitivities and specificities are shown in Tables 5.14 and 5.15. Assuming that all the probable cases were true cases, twenty-eight serum samples
(93.3%) had IgG4 antibodies against Toxocara using rTES-120, and this is similar to the sensitivity of rTES-30USM. Meanwhile 24 (80.0%) had IgG4 antibodies when tested with rTES-26 and rTES-32. Thus, an assay using rTES-120 seemed to be more sensitive compared to rTES-26 and rTES-32. One false-negative sample was from a patient who was clinically diagnosed as OLM (T3), and as expected this sample showed lower O.D value
(below the COV) than those obtained from patients with visceral disease. Another false- negative sample (T10) was a false which gave weak/low positive result when tested in IgG4-
ELISA using native antigen. However, both T3 and T10 were positive by assay using rTES-
30USM. Combination of results of rTES-30USM and TES-120 gave a sensitivity of 100%.
The specificity analysis of each IgG4 assay is shown in Table 5.13. As shown in
Table 5.15, the rTES-26, rTES-32, rTES-120 and rTES-30USM gave specificities between
92.0 – 96.2%, with most cross-reactions caused by STH (presumably false-positives). The rTES-26 showed greater specificity (96.2%) as compared to the specificities of rTES-32
(93.9%), rTES-120 (92.0%) and rTES-30USM (93.9%). None of the samples from healthy normal had IgG4 antibodies against T. canis.
In tables 5.16a and 5.16b, the PPV of 64-79% suggested that the assay developed was accurate and reliable, while the high NPV (95-99%) showed that the assay is capable of discriminating non-toxocara cases.
235 Table 5.12 Sensitivity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES- 30USM antigens
No Serum Code rTES-26 rTES-32 rTES-120 rTES-30USM ELISA ELISA ELISA ELISA 1 T1 0.145 0.156 0.408 0.312 2 T2 0.717 0.232 0.610 0.620 3 T3* 0.049 0.148 0.176 0.215 4 T4 0.550 1.358 0.588 0.796 5 T5 2.077 1.874 1.878 1.794 6 T6 0.251 0.299 0.444 1.624 7 T7 1.993 1.336 1.457 1.248 8 T8 0.432 1.331 0.562 0.256 9 T9 0.204 0.234 0.367 0.597 10 T10* 0.081 0.179 0.174 0.203 11 T12 3.490 0.339 2.706 0.482 12 T15 3.731 3.293 4.263 0.878 13 UMT#3 0.215 0.302 0.580 0.308 14 UMT#5 0.725 1.194 0.408 0.542 15 UMT#6 1.687 1.595 1.933 1.200 16 UMT#8 0.537 0.588 0.631 0.375 17 UMT#9 0.550 0.678 0.449 0.397 18 MBDR 60 0.054 0.076 0.488 0.040 19 MBDR 62 0.099 0.194 0.256 0.256 20 MBDR 64 0.392 0.426 0.281 0.655 21 MBDR 65 0.206 0.220 0.224 1.751 22 MBDR 66 0.231 0.230 0.229 0.223 23 MBDR 67 0.409 0.459 0.450 0.425 24 MBDR 68 1.225 1.008 1.149 0.756 25 T16 0.688 1.881 0.737 0.228 26 T17 0.682 0.545 0.845 0.231 27 96.41358.M 0.243 0.263 0.206 0.172 28 96.28371.H 0.584 2.563 0.964 1.256 29 98.29664.V 0.452 0.713 1.786 1.258 30 T18 0.145 0.168 0.559 0.180
ODs in bold and highlighted: false-negative results. Asterisk (*) indicating OLM cases.
236 Table 5.13 Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens No Serum Desc rTES-26 rTES-32 rTES-120 rTES- Code ELISA ELISA ELISA 30USM ELISA 1 PK 1 Healthy normal 0.051 0.054 0.125 0.001 2 PK 2 Healthy normal 0.095 0.128 0.067 0.011 3 PK 3 Healthy normal 0.050 0.085 0.068 0.006 4 PK 4 Healthy normal 0.069 0.082 0.085 0.021 5 PK 5 Healthy normal 0.009 0.002 0.016 0.040 6 PK 6 Healthy normal 0.065 0.129 0.004 0.010 7 PK 7 Healthy normal 0.060 0.021 0.025 0.020 8 PK 8 Healthy normal 0.102 0.003 0.033 0.060 9 PK 9 Healthy normal 0.085 0.003 0.038 0.030 10 PK 10 Healthy normal 0.025 0.071 0.035 0.040 11 PK 11 Healthy normal 0.013 0.068 0.076 0.040 12 PK 12 Healthy normal 0.015 0.023 0.010 0.010 13 PK 13 Healthy normal 0.003 0.025 0.069 0.037 14 PK 14 Healthy normal 0.001 0.141 0.162 0.106 15 PK 15 Healthy normal 0.002 0.122 0.065 0.060 16 PK 16 Healthy normal 0.015 0.010 0.029 0.030 17 PK 17 Healthy normal 0.023 0.102 0.115 0.050 18 PK 18 Healthy normal 0.004 0.027 0.073 0.006 19 PK 19 Healthy normal 0.007 0.015 0.030 0.042 20 PK 20 Healthy normal 0.007 0.020 0.063 0.044 21 PK 21 Healthy normal 0.012 0.069 0.080 0.059 22 PK 22 Healthy normal 0.010 0.014 0.009 0.045 23 PK 23 Healthy normal 0.011 0.020 0.014 0.049 24 PK 24 Healthy normal 0.109 0.174 0.140 0.121 25 PK 25 Healthy normal 0.037 0.079 0.071 0.054 26 PK 26 Healthy normal 0.024 0.056 0.065 0.036 27 PK 27 Healthy normal 0.008 0.044 0.001 0.013 28 PK 28 Healthy normal 0.002 0.020 0.042 0.023 29 PK 29 Healthy normal 0.032 0.043 0.005 0.011 30 PK 30 Healthy normal 0.040 0.198 0.040 0.041 31 PK 31 Healthy normal 0.040 0.028 0.051 0.028 32 PK 32 Healthy normal 0.005 0.041 0.054 0.014 33 PK 33 Healthy normal 0.061 0.110 0.042 0.048 34 PK 34 Healthy normal 0.034 0.107 0.017 0.047 35 PK 35 Healthy normal 0.074 0.063 0.054 0.036 36 PK 36 Healthy normal 0.137 0.103 0.090 0.047 37 PK 37 Healthy normal 0.017 0.020 0.027 0.018 38 PK 38 Healthy normal 0.039 0.098 0.027 0.018 39 PK 39 Healthy normal 0.024 0.030 0.012 0.007 40 PK 40 Healthy normal 0.019 0.028 0.025 0.013 41 PK 41 Healthy normal 0.041 0.077 0.031 0.027 42 PK 42 Healthy normal 0.036 0.038 0.031 0.000 43 PK 43 Healthy normal 0.033 0.099 0.022 0.016 44 PK 44 Healthy normal 0.113 0.092 0.073 0.041 45 PK 45 Healthy normal 0.030 0.085 0.028 0.008 46 PK 46 Healthy normal 0.032 0.046 0.036 0.014 47 PK 47 Healthy normal 0.014 0.003 0.012 0.006 48 PK 48 Healthy normal 0.014 0.056 0.026 0.010 49 PK 49 Healthy normal 0.009 0.001 0.015 0.005
237 Table 5.13(cont.) Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens
50 PK 50 Healthy normal 0.023 0.000 0.013 0.004 51 PK 51 Healthy normal 0.020 0.125 0.022 0.002 52 PK 52 Healthy normal 0.031 0.078 0.020 0.003 53 PK 53 Healthy normal 0.009 0.085 0.029 0.008 54 PK 54 Healthy normal 0.004 0.054 0.020 0.013 55 PK 55 Healthy normal 0.013 0.023 0.061 0.025 56 PK 56 Healthy normal 0.002 0.032 0.033 0.014 57 PK 57 Healthy normal 0.015 0.031 0.013 0.006 58 PK 58 Healthy normal 0.001 0.025 0.045 0.026 59 PK 59 Healthy normal 0.004 0.064 0.023 0.012 60 PK 60 Healthy normal 0.022 0.085 0.025 0.002 61 PK 61 Healthy normal 0.006 0.081 0.072 0.048 62 PK 62 Healthy normal 0.003 0.080 0.057 0.040 63 PK 63 Healthy normal 0.016 0.096 0.020 0.015 64 PK 64 Healthy normal 0.004 0.025 0.042 0.029 65 PK 65 Healthy normal 0.016 0.011 0.017 0.004 66 PK 66 Healthy normal 0.000 0.009 0.026 0.009 67 PK 67 Healthy normal 0.015 0.056 0.041 0.020 68 PK 68 Healthy normal 0.006 0.068 0.080 0.057 69 PK 69 Healthy normal 0.016 0.079 0.033 0.035 70 PK 70 Healthy normal 0.021 0.005 0.024 0.026 71 PK 71 Healthy normal 0.005 0.017 0.095 0.056 72 PK 72 Healthy normal 0.029 0.013 0.023 0.001 73 PK 73 Healthy normal 0.010 0.079 0.023 0.004 74 PK 74 Healthy normal 0.024 0.117 0.021 0.028 75 PK 75 Healthy normal 0.030 0.045 0.036 0.034 76 PK 76 Healthy normal 0.027 0.035 0.024 0.050 77 PK 77 Healthy normal 0.034 0.006 0.019 0.036 78 PK 78 Healthy normal 0.040 0.038 0.019 0.043 79 PK 79 Healthy normal 0.035 0.006 0.030 0.038 80 PK 80 Healthy normal 0.026 0.048 0.035 0.003 81 PK 81 Healthy normal 0.062 0.033 0.033 0.034 82 PK 82 Healthy normal 0.005 0.065 0.038 0.052 83 PK 83 Healthy normal 0.056 0.002 0.032 0.045 84 PK 84 Healthy normal 0.070 0.001 0.023 0.038 85 PK 85 Healthy normal 0.059 0.003 0.038 0.051 86 PK 86 Healthy normal 0.025 0.012 0.045 0.055 87 PK 87 Healthy normal 0.007 0.058 0.037 0.060 88 PK 88 Healthy normal 0.003 0.046 0.046 0.050 89 PK 89 Healthy normal 0.033 0.006 0.018 0.088 90 PK 90 Healthy normal 0.057 0.033 0.011 0.001 91 PK 91 Healthy normal 0.011 0.056 0.024 0.011 92 PK 92 Healthy normal 0.042 0.087 0.025 0.016 93 PK 93 Healthy normal 0.052 0.077 0.039 0.062 94 PK 94 Healthy normal 0.041 0.006 0.014 0.017 95 PK 95 Healthy normal 0.039 0.008 0.032 0.007 96 PK 96 Healthy normal 0.023 0.003 0.019 0.025 97 PK 97 Healthy normal 0.023 0.016 0.023 0.017 98 PK 98 Healthy normal 0.022 0.008 0.022 0.008 99 PK 99 Healthy normal 0.008 0.059 0.018 0.009 100 PK 100 Healthy normal 0.031 0.015 0.031 0.036
238 Table 5.13(cont.) Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens
101 SB58 Mf+ve 0.092 0.100 0.112 0.130 102 SB21 Mf+ve 0.021 0.021 0.093 0.063 103 SB17 Mf+ve 0.030 0.073 0.013 0.186 104 SB1 Mf+ve 0.380 1.100 0.790 0.825 105 SB6 Mf+ve 0.505 1.205 0.697 0.866 106 SB20 Mf+ve 0.159 0.128 0.152 0.100 107 SB54 Mf+ve 0.085 0.153 0.119 0.154 108 SB59 Mf+ve 0.023 0.025 0.118 0.127 109 SB52 Mf+ve 0.133 0.160 0.165 0.129 110 SB26 Mf+ve 0.001 0.129 0.152 0.034 111 SB14 Mf+ve 0.115 0.127 0.168 0.122 112 SB62 Mf+ve 0.060 0.085 0.132 0.124 113 SB30 Mf+ve 0.107 0.299 0.214 0.267 114 SB25 Mf+ve 0.045 0.096 0.175 0.130 115 SB12 Mf+ve 0.032 0.074 0.082 0.077 116 SB28 Mf+ve 0.125 0.034 0.191 0.104 117 SB10 Mf+ve 0.003 0.092 0.092 0.179 118 SB36 Mf+ve 0.014 0.117 0.031 0.117 119 SB32 Mf+ve 0.134 0.178 0.189 0.164 120 SB45 Mf+ve 0.030 0.077 0.075 0.067 121 SB46 Mf+ve 0.002 0.038 0.111 0.138 122 SB55 Mf+ve 0.058 0.056 0.104 0.111 123 SB16 Mf+ve 0.034 0.002 0.058 0.034 124 SB76 Mf+ve 0.073 0.190 0.175 0.088 125 SB13 Mf+ve 0.169 0.151 0.104 0.125 126 SB11 Mf+ve 0.083 0.096 0.121 0.160 127 SB83 Mf+ve 0.004 0.076 0.062 0.031 128 WB56 Mf+ve 0.013 0.095 0.195 0.030 129 TP 1 Toxoplasmosis 0.001 0.072 0.112 0.018 130 TG 1 Toxoplasmosis 0.001 0.149 0.152 0.020 131 UMT70 Toxoplasmosis 0.089 0.117 0.090 0.130 132 UMT45 Toxoplasmosis 0.065 0.088 0.092 0.076 133 UMT66 Toxoplasmosis 0.052 0.024 0.092 0.149 134 UMT90 Toxoplasmosis 0.064 0.104 0.069 0.107 135 UMT6 Toxoplasmosis 0.090 0.029 0.036 0.007 136 UMT30 Toxoplasmosis 0.100 0.007 0.043 0.072 137 UMT21 Toxoplasmosis 0.094 0.024 0.034 0.149 138 UMT88 Toxoplasmosis 0.087 0.095 0.047 0.185 139 UMT49 Toxoplasmosis 0.128 0.028 0.042 0.170 140 UMT78 Toxoplasmosis 0.412 0.352 0.459 0.425 141 UMT35 Toxoplasmosis 0.372 0.270 0.512 0.614 142 UMT87 Toxoplasmosis 0.068 0.082 0.064 0.104 143 UMT79 Toxoplasmosis 0.031 0.125 0.122 0.099 144 UMT29 Toxoplasmosis 0.018 0.149 0.025 0.036 145 UMT72 Toxoplasmosis 0.046 0.028 0.009 0.050 146 UMT34 Toxoplasmosis 0.037 0.093 0.112 0.170 147 UMT71 Toxoplasmosis 0.089 0.045 0.138 0.189 148 UMT60 Toxoplasmosis 0.062 0.022 0.093 0.136 149 A28 Amoebiasis 0.036 0.003 0.041 0.052 150 EH74 Amoebiasis 0.162 0.136 0.166 0.157
239 Table 5.13(cont.) Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens 151 EH79 Amoebiasis 0.040 0.089 0.002 0.117 152 EH10 Amoebiasis 0.027 0.289 0.265 0.518 153 EH18 Amoebiasis 0.031 0.125 0.113 0.104 154 EH70 Amoebiasis 0.108 0.190 0.182 0.169 155 EH47 Amoebiasis 0.154 0.125 0.399 0.048 156 EH53 Amoebiasis 0.021 0.053 0.076 0.156 157 EH76 Amoebiasis 0.035 0.046 0.382 0.100 158 EH1 Amoebiasis 0.297 0.518 0.273 0.444 159 EH27 Amoebiasis 0.014 0.142 0.009 0.045 160 EH77 Amoebiasis 0.053 0.069 0.128 0.085 161 EH9 Amoebiasis 0.185 0.184 0.072 0.150 162 EH8 Amoebiasis 0.010 0.082 0.038 0.091 163 EH23 Amoebiasis 0.127 0.025 0.002 0.078 164 EH26 Amoebiasis 0.028 0.06 0.098 0.098 165 EH46 Amoebiasis 0.071 0.162 0.444 0.087 166 EH31 Amoebiasis 0.018 0.057 0.061 0.104 167 EH59 Amoebiasis 0.060 0.151 0.316 0.129 168 EH5 Amoebiasis 0.056 0.290 0.125 0.357 169 EH55 Amoebiasis 0.006 0.107 0.035 0.070 170 EH66 Amoebiasis 0.029 0.072 0.029 0.184 171 A64 Amoebiasis 0.117 0.025 0.012 0.079 172 EH85 Amoebiasis 0.009 0.032 0.045 0.036 173 A32 Amoebiasis 0.099 0.146 0.632 0.145 174 EH60 Amoebiasis 0.002 0.032 0.038 0.050 175 A39 Amoebiasis 0.098 0.326 0.091 0.454 176 A49 Amoebiasis 0.238 0.256 0.154 0.342 177 EH56 Amoebiasis 0.126 0.125 0.099 0.093 178 EH11 Amoebiasis 0.012 0.017 0.035 0.167 179 Tri 1 STH 0.042 0.060 0.190 0.086 180 Tri 2 STH 1.115 0.148 0.307 0.065 181 Tri 5 STH 0.114 0.128 0.020 0.022 182 Tri 6 STH 0.077 0.025 0.013 0.122 183 Tri 7 STH 0.046 0.067 0.002 0.184 184 Tri 8 STH 0.041 0.899 1.289 0.996 185 Tri 11 STH 0.046 0.099 0.036 0.045 186 Tri 12 STH 0.078 0.064 0.023 0.065 187 Asc 1 STH 0.001 0.007 0.010 0.021 188 Asc 2 STH 0.006 0.023 0.036 0.031 189 Asc 3 STH 0.026 0.168 0.147 0.112 190 Asc 5 STH 0.098 0.148 0.125 0.045 191 Asc 6 STH 0.051 0.055 0.066 0.098 192 Asc 7 STH 0.022 0.041 0.052 0.103 193 Asc 10 STH 0.168 0.099 0.122 0.060 194 Asc 12 STH 0.001 0.016 0.034 0.100 195 Mix 1 STH 0.025 0.003 0.065 0.029 196 Mix 5 STH 0.052 0.120 0.304 0.125 197 Mix 9 STH 0.055 0.092 0.067 0.124 198 Mix 10 STH 0.009 0.022 0.047 0.081 199 Mix 13 STH 0.036 0.010 0.083 0.018 200 Mix 14 STH 0.020 0.008 0.085 0.035 201 Mix 16 STH 0.014 0.044 0.047 0.058
240 Table 5.13(cont.) Specificity study of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens
202 Mix 17 STH 0.158 0.091 0.065 0.171 203 Mix 18 STH 0.020 0.018 0.007 0.093 204 Mix 19 STH 0.100 0.123 0.008 0.105 205 Nec 2 STH 0.008 0.050 0.024 0.053 206 Nec 4 STH 0.012 0.745 0.322 0.950 207 STG 1 Strongyloidiasis 0.011 0.005 0.021 0.030 208 STG 2 Strongyloidiasis 0.279 0.480 0.582 0.857 209 STG 3 Strongyloidiasis 0.085 0.098 0.045 0.120 210 STG 4 Strongyloidiasis 0.025 0.056 0.036 0.161 211 STG 5 Strongyloidiasis 0.011 0.069 0.025 0.130 212 Gnat 1 Gnathostomiasis 0.025 0.060 0.052 0.100
ODs in bold and highlighted: false-positive results.
241 Table 5.14 Summary of sensitivity evaluations of IgG4-ELISA using rTES-26, rTES-32, rTES-120 and rTES-30USM
IgG4-ELISA Total Positive Negative Sensitivity (%) rTES-26 30 24 6 80.0 rTES-32 30 24 6 80.0 rTES-120 30 28 2* 93.3 rTES-30USM 30 28 2* 93.3 Sensitivity of IgG4-ELISA using rTES-26/32 vs rTES-120/30USM was statistically significant (p <0.001)
* 2 serum samples which were negative by rTES-30USM gave positive reactions by rTES-120 and vice-versa. Thus, combination of rTES-30USM and rTES-120 gave sensitivity of 100%.
Table 5.15 Summary of specificity evaluations of IgG4-ELISA using rTES-26, rTES- 32, rTES-120 and rTES-30USM Type of sera Negative by Negative by Negative by Negative by rTES-26 rTES-32 rTES-120 rTES-30USM ELISA ELISA ELISA ELISA
A.lumbricoides, 27/28 (1) 26/28 (2) 24/28 (4) 26/28 (2) T. trichiura, hookworm
S. stercoralis 4/5 (1) 4/5 (1) 4/5 (1) 4/5 (1)
G. spinigerum 1/1 (0) 1/1 (0) 1/1 (0) 1/1 (0)
E. histolytica 28/30 (2) 25/30 (5) 23/30 (7) 25/30 (5)
B. malayi 26/28 (2) 25/28 (3) 25/28 (3) 25/28 (3) microfilaremic
T. gondii 18/20 (2) 18/20 (2) 18/20 (2) 18/20 (2)
Healthy 100/100 (0) 100/100 (0) 100/100 (0) 100/100 (0) individuals Total 204/212 (8) 199/212 (13) 195/212 (17) 199/212 (13) Specificity Specificity Specificity Specificity (96.2%) (93.9%) (92.0%) (93.9%)
Specificity showed included 100 healthy individuals. * The number in parenthesis denotes false positive results.
Table 5.16a Specificity, accuracy, PPV and NPV of rTES ELISA using monoclonal mouse anti-human IgG4 antibody (include 30 healthy normals)
242 IgG4-ELISA Specificity (%) Accuracy (%) PPV (%) NPV (%) rTES-26 94.4 (134/142) 92.5 (172/186) 78.9 (30/38) 95.9 (142/148) rTES-32 90.8 (129/142) 90.0 (172/191) 69.8 (30/43) 95.9 (142/148) rTES-120 88.0 (125/142) 90.0 (172/191) 63.8 (30/47) 98.6 (142/144) rTES-30USM 90.8 (129/142) 92.0 (172/187) 69.8 (30/43) 98.6 (142/144)
Table 5.16b Specificity, accuracy, PPV and NPV of rTES ELISA using monoclonal mouse anti-human IgG4 antibody (exclude healthy normals)
IgG4-ELISA Specificity (%) Accuracy (%) PPV (%) NPV (%) rTES-26 92.9 (104/112) 91.0 (142/156) 78.9 (30/38) 94.9 (112/118) rTES-32 88.4 (99/112) 88.2 (142/161) 69.8 (30/43) 94.9 (112/118) rTES-120 84.8 (95/112) 88.2 (142/161) 63.8 (30/47) 98.2 (112/118) rTES-30USM 88.4 (99/112) 90.4 (142/157) 69.8 (30/43) 98.2 (112/114) PPV : positive predictive value NPV : negative predictive value
5.3.5 Statistical analysis
The differences in sensitivities between rTES-26/rTES-32 and rTES-30USM/rTES-120 were highly statistically significant (p<0.001) as determined by Chi-square analysis. However, as shown in Table 5.17, One-Way ANOVA analysis demonstrated no significant differences
(p>0.05) between mean O.Ds obtained from 30 sera from Toxocara patients between all groups.
243 The rTES-26 was shown to have higher specificity as compared to that of rTES-32, rTES-120 and rTES-30USM (rTES-26>rTES-32/rTES-30USM>rTES-120). However, there was marginally no significant difference between the specificities of rTES-26 and rTES-120
(p=0.059), between rTES-26 and rTES-30USM/rTES-32 or between rTES-30USM/rTES-32 and rTES-120. Since in terms of specificity the rTES-32 was not shown to be better than rTES-26 and have similar sensitivity as rTES-26, the rTES-32 was excluded from the final assay for diagnosis of toxocariasis.
Table 5.17 One-Way ANOVA analysis of the comparison of mean O.Ds of IgG4 assays on 30 toxocariasis serum samples using rTES-26, rTES-32, rTES-120 and rTES-30USM antigens
Std. F stata (df) p-value IgG4-ELISA Mean Deviation
rTES-26 0.761 0.940 0.388 (3, 116) 0.762 rTES-32 0.796 0.800 rTES-120 0.860 0.892
244 rTES-30USM 0.642 0.507 a ANOVA test df degree of freedom There is no significant difference between the four tests as indicated by the p-value (sig.) of >0.05 (0.762)
Table 5.18 Pearson Chi-Square analysis of the comparison of specificity of IgG4 assays using rTES-26, rTES-32, rTES-30USM and rTES-120 antigens
include 30 healthy exclude healthy normals (p-value) normal (p-value) rTES-26 & rTES-30USM/rTES-32 0.257 0.252 rTES-30USM/rTES-32 & rTES-120 0.440 0.433 rTES-26 & rTES-120 0.059 0.056 Asymp. sig (2-sided) p<0.05 p<0.05
There was marginally no significant difference between the specificities of rTES-26 and rTES-120, between rTES-26 and rTES-30USM/rTES-32 or between rTES-30USM/rTES-32 and rTES-120.
5.4 Discussion
Many assays have been developed to diagnose Toxocara infections. This depends mostly on serological tests to detect the presence of specific anti-Toxocara antibodies because eggs are not produced and larvae are not eliminated by humans. The use of ELISA in the serodiagnosis of toxocariasis has been shown to be of great value (de Savigny et al., 1979).
TES antigens derived from L2 larvae maintained in defined medium in vitro have been extensively used for the immunodiagnosis of human toxocariasis. However, not all TES
245 antigens are specific. Many serum samples from patients with ascariasis, filariasis, strongyloidiasis, gnathostomiasis and schistosomiasis showed cross-reactivities with TES antigens by ELISA and Western blot (Magnaval et al., 1991; Camargo et al., 1992; Rahmah et al., 2005). Since nematodes are known to share common antigenic components, the use of
TES crude antigen in tropical areas where multiparasitism is common will be problematic. In most areas in which STH especially A. lumbricoides is endemic, co-infection with ascaris and toxocara is likely to be sufficiently common to confuse serodiagnosis (Nunes et al.,
1999). Cross-reactivity in antigen-antibody reaction is thus an important problem and of great concern in the serodiagnosis of parasitic infections, especially in the tropics (Lokman
Hakim et al., 1997).
At present, diagnosis of human toxocariasis by commercial kits is performed by detecting IgG in the patient’s serum, in an ELISA format. However, these IgG-assays lacked sufficient specificity since they give false-positive reactions with other parasitic helminth infections that are commonly found in tropical countries, and thus pose a significant diagnostic challenge (Magnaval et al., 2001). These IgG-assays are useful only in differential diagnosis, but when the results are positive, the test interpretation can be problematic (Rahmah et al., 2005). Thus, the development of a reliable assay to demonstrate the presence of specific antibodies against Toxocara is a need towards improving diagnosis of the disease.
T. canis infection has been reported to induce T helper 2 systemic immune responses, with the production of the antibodies IgG1 and IgE (Kuroda et al., 2001). The eosinophilia and increased IgE concentration are due to increased numbers and activity of T cells with a pattern of secretion of cytokines (IL-4 and IL-5) and a reduction in secretion of
T helper 1 lymphocytes whilst IL-4 amplifies production of IgE (Buijs et al., 1997).
Figuiredo et al. (2005) have observed extremely significant associations between positive serology for T. canis with elevated IgE levels (above 200 IU/dl) and eosinophilia (above 400 cells/mm3). VLM patients had been reported to have 10-15 times the average normal level of
IgE as determined by the radioimmunosorbent test (Hogarth-Scott et al., 1969).
246 Determination of serum total IgE is thus very helpful in the diagnosis of toxocariasis. High levels of total IgE in toxocariasis patients are indicative or supportive of true positive cases.
Thus at the beginning of the study, serum samples from Toxocara infections, other related infections and normal individuals were screened for total IgE. The results obtained in this study showed that 21 out of 25 (84%) of Toxocara patients have increased levels of total
IgE, thus demonstrating that most Toxocara positive cases have significant levels of total serum IgE. This is consistent with the previous reports of elevated total IgE in toxocariasis patients (Kuroda et al., 2001; Moreira-Silva et al., 2002; Figueiredo et al., 2005). However, the results in this study also showed that non-toxocara infections and healthy normals also demonstrated high total serum IgE, e.g. STH (15/28), B. malayi microfilaremics (9/15), amoebiasis (7/12), toxoplasmosis (4/12), strongyloidiasis (3/5) and healthy normals (21/68) showed IgE titer of more than 100 IU/ml. High serum total IgE was also seen in some seronegative individuals (Giacometti et al., 2000). Neverthesless Chi-square analysis showed that serum samples of Toxocara patients had significantly greater numbers of positive cases
(p<0.001) as compared to other groups. The observations that some individuals without toxocariasis also had high levels of total IgE were not surprising since total IgE level have also been shown to increase in allergy and other parasitic/nematode infections and is well correlated with the presence of larva in tissues.
In chapter 4, rTES-26, rTES-32 and rTES-120 recombinant proteins were shown to be immunologically reactive and specific by Western blot analysis. The purified recombinant antigens were employed in developing immunoassays that were specific and sensitive for detection of toxocariasis. Optimizations were performed with various concentrations of antigens and dilutions of primary (patients’ sera) and secondary antibodies.
Excessive amounts of primary and secondary antibodies were avoided to minimize the background problems and wastage of antibodies. In this study, the comparison between PBS and blocking solution as a diluent for primary and secondary antibodies was not done since the PBS gave negligible background readings. The optimum conditions were important to provide a clear discrimination of positive and negative samples. Subsequently, laboratory
247 evaluations were carried out to determine the sensitivities and specificities of each assay. A panel of patients’ sera comprised Toxocara infections, other parasitic related infections and healthy individuals were evaluated.
High levels of IgE specific for TES have also been reported in the serum samples of patients with clinical signs suggestive of Toxocara infection (Smith & Kennedy, 1993;
Smith, 1998). IgE antibody has also been shown to increase the specificity of the assays
(Wiechinger, 1998). However, not all patients with elevated total IgE levels demonstrated specific anti-Toxocara IgE. In a study performed by Tay (1999), only two of seven
Toxocara patients had an increased serum IgE level. Pawlowski (2001) reported Toxocara- specific IgE was present in half of the patients. The results of the IgE assay developed in this study were in agreement with that of Pawlowski (2001,) whereby only half of Toxocara patients had detectable Toxocara-specific IgE antibodies. There were also other studies which showed that in human toxocariasis, total serum IgE levels were significantly increased, whereas Toxocara-specific IgE was not detected or was at a very low level
(Uhlikova et al., 1996).
The level of IgM antibody has also been observed to be elevated in toxocariasis patients (Takamoto et al., 1998; Cuellar et al., 2001; Reiterova et al., 2003). There are reports that acute infection is best made by the demonstration of raised Toxocara-specific
IgM (Shetty & Aviles, 1999). However, only a few studies identified the usefulness of specific IgM for Toxocara serodiagnosis. In this study, IgM was shown not to be a good marker for Toxocara infection since only one sample was positive by this assay. This result may be expected because toxocariasis is usually a chronic disease.
The sensitivity and specificity of TES-ELISA was reported to be significantly improved by the use of IgG subclass antibodies (Obwaller et al., 1998; Rahmah et al., 2005;
Norhaida et al., 2008; Watthanakulpanich et al., 2008). Human IgG consists of four subclasses (IgG1 – 4) that are characterized by antigenic differences in their heavy chains.
IgG1-4 constitutes approximately 61, 30, 5 and 4% of total IgG, respectively. Each IgG subclass has different biological and physiochemical properties and hence may be
248 preferentially produced in response to different antigens (Watthankulpanich et al., 2008).
IgG1 and IgG3 were reported to be produced in response to protein antigens whilst IgG2 responses was reported to be directed primarily against carbohydrate antigens (which are common components of TES) and IgG4 reflects prolonged antigenic stimulation (Scott et al.,
1990).
In this study, these purified recombinant proteins were also used to develop immunoassays to detect specific antibodies to the IgG subclasses (IgG1-IgG4). Neither the sensitivity nor specificity achieved by IgG subclasses, IgM and IgE was acceptable. IgG4-
ELISA assay was found to demonstrate the least cross reactivity, and hence the highest specificity while IgG1-ELISA demonstrated the lowest specificity. The decreasing order of the assay specificity was as follows: IgG4>IgG3>IgG2>IgG1 (Table 5.12). IgG4 also gave the greatest sensitivity as compared to other IgG subclasses, the decreasing order of the assay sensitivity were as follows: IgG4>IgG2>IgG1≥IgG3. These results obtained were in contrast with the study done by Watthanakulpanich et al. (2008). They reported that all four
IgG subclass antibodies gave approximately 10-fold increases in ELISA O.D values for toxocariasis patients compared to healthy normals. Anti-Toxocara IgG2 gave the greatest sensitivity (98%) (IgG2>IgG3>IgG4>IgG1>IgG) while anti-Toxocara IgG3 gave the greatest specificity (81%) (IgG3>IgG1>IgG>IgG2 and IgG4). The differences in the results of the study by Watthanakulpanich et al. (2008) from the present study are probably due to the fact that native TES antigen was employed in their study while recombinant antigens were employed in our study. In another study the predominant IgG subclass in toxocariasis patients with VLM, OLM or asymptomatic individuals was IgG1, followed by IgG2, IgG4 and IgG3 (Obwaller et al, 1998).
The result of the present study was in agreement with the previous studies which reported the significant increase of specificity for detection of toxocariasis with IgG4 antibody as compared to total IgG (Wiechinger, 1998; Rahmah et al., 2005; Norhaida et al.,
2008). In this respect it is similar to the situation with lymphatic and non-lymphatic filariasis whereby elevated IgG4 levels have been associated with active infection in other helminthic
249 infections such as filariasis and onchocerciasis (Kwan Lim et al., 1990; Weil et al., 1990;
Rahmah et al., 2001) and has been used as a marker of active infection (Kwan Lim et al.,
1990; Weil et al., 2000; Rahmah et al., 2001; Klion et al., 2003). Immunological responses in patients chronically infected with filariasis revealed the presence of very prominent IgG4 responses to parasite antigen (Ottesen et al., 1985), which were found to induce very strong
IgG4 response. Similarly, IgG4 response was predominant in non-filarial infections such as echinococcosis, neurocysticercosis and sparganosis (Grimm et al., 1998; Yang et al., 1998;
Chung et al., 2000). IgG4 responses to TES might reflect ongoing infection with migrating larvae (Rahmah et al., 2005) and prolonged antigenic stimulation (Scott et al., 1990). Since high levels of IgE and IgG4 are produced during helminth infection, IgG4 antibodies are said to mask the presence of IgE by a competitive mechanism toward the same epitopes of the parasite. In fact, IgE antibody titers increased after depletion of IgG from serum, due to the antibody blocking effect (Magnaval et al., 2002). However, until now no detection test based on IgG4 is available commercially for the detection of Toxocara infection.
Based on the above results, it was evident that only the assay based on IgG4 antibody displayed good specificity, thus subsequently this was employed as the secondary antibody of choice for the final development of diagnostic assay for toxocariasis. The diagnostic utility of each purified recombinant antigen and rTES-30USM (previously produced in our laboratory, Norhaida et al., 2008) were further evaluated by IgG4-ELISA assay using 242 serum samples which included 30 sera from patients with clinical, haematological and serological evidence of toxocariasis. Out of these, 93.3% (28/30) were reactive for anti-toxocara IgG4 antibodies in IgG4 assays employing rTES-120 and rTES-
30USM and 80.0% (24/30) sensitivities for both rTES-26 and rTES-32 IgG4-ELISA. Thus rTES-120/rTES-30USM antigen was more sensitive (93.3%) than assays using either rTES-
26 or rTES-32 antigens, and the combination of results of rTES-30USM and rTES-120 gave
100% sensitivity. One false-negative sample from a patient with OLM (T3) examined in this study showed a lower O.D value (below the COV) than those obtained from patients with visceral disease. This is not surprising since it has been reported that the sensitivity of
250 ELISA for ocular toxocariasis is generally much lower than that for visceral toxocariasis.
This might be due to differences in the immune responses in the eye and liver and may also be related to the number of T. canis larvae trapped in the eye or to the longer period between the onset of illness and serologic testing (Schantz, 1989). However, comparison of the mean
O.Ds obtained from 30 toxocariasis serum samples showed that no significant difference in sensitivities (p>0.05) were observed between assays.
The rTES-26, rTES-32, rTES-120 and rTES-30USM gave specificities between 92.0 to 96.2%. Of the 100 sera from healthy blood donors that were tested, all of them were found to be negative in all assays. One explanation for the high specificity of these recombinant antigens is that it is a single or homogenous molecule, while native TES consists of multiple components with a wide range of molecular masses. Second, in contrast with glycosylated nature of native TES (Page et al., 1992), the recombinant antigen produced in bacteria is not glycosylated. Thus this may lead to a great reduction in cross-reactivities with antibodies that recognized the sugar moietis of the TES produced by T. canis larvae (Maizels et al., 1987).
The absence of significant cross-reactivity in these sera confirms the high specificity of these recombinant antigens in detecting anti-toxocara antibodies and its usefulness in a tropical region like Malaysia, where multiple helminthic infections are common.
As expected, most false positives obtained in this study were caused by STH since environmental contamination with Toxocara and STH are common in the tropics. Thus
Toxocara co-infections among the false positive non-toxocara samples are very probable. At p<0.05, there was marginally no significant difference between the specificities of rTES-26 and rTES-120 (p=0.059), between rTES-26 and rTES-30USM or between rTES-30USM and rTES-120.
In the present study, the low molecular weight band (26 kDa) appeared to be more specific than higher molecular weight band (120 kDa). This is consistent with the results of previous studies in which low molecular weight bands (24 – 35 kDa) of native TES were shown to be specific for toxocariasis, while the high molecular weight TES bands showed reactivity with sera from various helminth infections (Magnaval et al., 1991; Park et al.,
251 2000). This could explain the pattern of specificity rates obtained in this study (rTES-26 > rTES-30USM/32 > rTES-120).
The high PPV suggested reliability and accuracy of the test developed. In addition the high NPV showed that the test is capable of discriminating non-toxocara cases. Since in terms of specificity the rTES-32 was not shown to be better than rTES-26 and have similar sensitivity as rTES-26, the rTES-32 was excluded for the final assay for diagnosis of toxocariasis. Instead the previously produced rTES-30USM was included in the final IgG4- assay for toxocariasis due to its high sensitivity and the observation that the combination of results of rTES-30USM and rTES-120 produce 100% sensitivity.
Therefore, when the assay with all three recombinant antigens (rTES-26, rTES-
30USM and rTES-120) gave positive results and the clinical symptoms are consistent with
Toxocara infection, there is strong evidence/high likelihood that it is a true positive case.
The same conclusion applies when combination of rTES-26 and rTES-120 ELISA or rTES-
26 and rTES-30USM ELISA are positive. When only rTES-30USM or rTES-120 ELISA is positive, although the likelihood is not as high for a true positive case nevertheless there is good evidence that the patient was infected.
In this study, the recombinant antigens were not mixed together and separately coated onto ELISA wells. This is based on our previous experience with development of
ELISA for lymphatic filariasis whereby combining recombinant antigens appeared to decrease the sensitivities for the detection of infection, probably because of the reduced availability of antigen binding sites in the wells as compared to that available when only one kind of recombinant antigen was employed (Rohana et al., 2007).
This study demonstrated that utilization of rTES-26, rTES-30USM and rTES-120 recombinant antigens with detection of specific anti-toxocara IgG4 antibody enabled the development of a highly specific and sensitive test for toxocariasis.
252 CHAPTER 6
SUMMARY AND CONCLUSION
Toxocariasis, caused by infective larvae of Toxocara species is one of the neglected tropical diseases (NTDs) (Hotez, 2007; 2008) which constitutes a public health problem and has been reported in many countries. In Malaysia, studies on various populations have shown significant anti-Toxocara antibody seroprevalence of 11-58% (Lokman Hakim et al., 1992,
1993, 1997; Patrick et al., 2001). T. canis, which affects dogs and other canines is the agent
253 most commonly implicated in the etiology of this illness among human population and is often associated with pica and poor hygiene (Fisher, 2003). In humans, when Toxocara eggs containing infective larvae are accidentally ingested, the larvae hatch in the small intestine and migrate through somatic organs, usually the liver and eyes. It commonly presents as visceral larva migrans, ocular larva migrans and covert toxocariasis. Worryingly, infection may involve invasion of the brain. During its migration through the host tissues, the larva induces humoral immune responses characterized by increased levels of IgG, IgM and IgE
(Elephant et al., 2006).
In human beings, the diagnosis of this disease is problematic since the clinical symptoms are unspecific and the larval stage of T. canis cannot be directly detected from regular clinical samples such as faeces or blood. Since humans are the paratenic hosts and the larvae do not complete their development, they do not arrive at the stage of egg deposition, hence making it impossible for direct diagnosis. Therefore, examination of stools has no role in the evaluation of toxocariasis. However, the presence of Ascaris and Trichuris eggs in faeces, indicating faecal exposure, increases the probability of Toxocara larvae in the tissues. Definitive diagnosis of toxocariasis is by histopathological examination and identification of Toxocara larvae from biopsy tissues. However it is very difficult to acquire the biopsy samples and is time-consuming. The determination of total IgE may also assist in diagnosis as shown in this study whereby significantly more (p<0.001) serum samples from
Toxocara patients had elevated total IgE compared to other groups. In view of the above difficulties in toxocara diagnosis, the detection of anti-toxocara specific antibodies in serum or other biological fluids using immunoassays is the only reliable method of diagnosis.
Currently, the most common serology tests being used are commercial IgG-ELISA kits, which employ native TES antigen obtained from in vitro culture of T. canis second- stage (L2) larvae (Smith & Rahmah, 2006). Prior to the use of TES as a diagnostic antigen, most surveys of human infection were based on tests using water soluble, somatic antigens derived from T. canis embryonated eggs, larvae or adults. Most of these non-TES antigens lacked sensitivity and possessed a high degree of cross-reactivity with Ascaris and other
254 ascarid antigens (Smith, 1998). Unlike worm somatic antigen, TES is released from living larvae, thus it is likely to enter the blood and potentially provide a quantitative and qualitative marker for active infection.
However, use of native TES antigen for serodiagnosis of human toxocariasis has some drawbacks. Studies using SDS-PAGE and Western blotting revealed that native TES antigens comprised a complex mixture of antigens, some of which are responsible for cross- reactions observed with sera from patients suffering from various non-toxocaral diseases.
The cross reactions between Toxocara and other geohelminth antigens (A. lumbricoides, T. trichiura, hookworm and S. stercoralis) made the assays difficult to interpret. This is of great concern in the tropics where polyparasitism is very common, thus the specificity is often too low especially to be used in developing countries where geohelminthic infections are common (Lynch et al., 1988; Lokman Hakim et al., 1997; Rahmah et al., 2005) and co- infections of Ascaris and Toxocara can confuse the serodiagnosis (Nunes et al., 1999). This is not surprising since their close taxonomic relationships cause them to share common antigens and epitopes on the secreted antigens of infective larvae. These include metabolic products of the worms which are antigenic and stimulate the production of cross-reactive antibodies. In addition, many serum samples from patients with filariasis, gnathostomiasis and schistosomiasis also showed significant reactivity with TES antigens (Magnaval et al.,
1991; Camargo et al., 1992). Production of TES antigen is also limited by the capacity of parasites culture, is time-consuming and laborious (Smith & Rahmah, 2006; Alcantara-
Neves et al., 2008).
The relatively low specificity of the present diagnostic kits for toxocariasis had called for alternative diagnostic methods. ELISA using recombinant T. canis antigen is highly recommended in tropical and subtropical regions where various parasitic infections are commonly endemic (Coelho et al., 2005). The single or homogenous nature of a recombinant antigen contributes to its high specificity, as compared to TES which consists of multiple components with a wide range of molecular masses. Secondly, the recombinant
255 antigen produced in bacteria is not glycosylated in contrast to glycosylated native TES (Page et al., 1992). The glycosylation may also lead to an increase in cross-reaction with antibodies that recognize the sugar moieties of the TES produced by T. canis larvae (Maizels et al.,
1987).
Yamasaki et al. (2000) reported the development of a highly specific recombinant T. canis antigen (rTES-30) which yielded greater specificity as compared to the native TES antigen. This recombinant antigen showed high sensitivity and specificity for detection of toxocariasis in IgG-ELISA (Lim et al., 1999; Yamasaki et al., 2000; Coelho et al., 2005).
The recombinant antigen was also previously produced in our laboratory by assembly PCR
(rTES-30USM) and was shown, by IgG4-ELISA, to be highly specific and sensitive for detection of toxocariasis (Norhaida et al., 2008). Another recombinant antigen, rTES-120 has also been reported to have diagnostic potential for toxocariasis since it has been shown to be sensitive and specific as a diagnostic reagent when employed in Western blot assay using IgG antibody (Fong et al., 2003; 2004). However, only eight cases of toxocariasis were tested by the rTES-120 IgG assay and no further validation studies were done to evaluate its potential for diagnostic value.
To date, rTES-26 and rTES-32 proteins-based assays have not been reported for
Toxocara serodiagnosis. However, a 32 kDa band has been reported to react with serum samples from patients, mice and rabbits infected with T. canis in Western blot analysis using native TES antigen (Kennedy et al., 1987). Moreover, the appearance of protein bands at 26 and 32 kDa are being used as two of six serodiagnostic markers for confirmation of toxocariasis in a commercial Western blot IgG kit which employ native TES antigen
(Testline, USA). Although previously produced rTES-26 was reported to have poor reactivity to human infection sera (Gems et al., 1995), based on its use in the commercial
Western blot kits,we suspected that the poor reactivity may be due to the different expression and purification systems employed. We thus hypothesized that TES-26 would be a useful candidate for this study, along with TES-32 and TES-120. Therefore, the ORF of the genes
256 which encode TES-26, TES-32 and TES-120 were selected for this study in our effort to develop a good diagnostic assay for human toxocariasis.
Approximately 90 stray dogs and 20 puppies were collected and sacrificed from
September 2004 to August 2005, from three different states in Malaysia. These animals were available because of the government’s policy to eliminate animals which no longer have owners, since they may be carriers of diseases like rabies. Moreover this will also address the problem of increased incidence of dog attacks on people. Enormous challenges were faced during the sample collections, due to logistics issues and the fact that tapeworm (rather than T. canis) was the most common infection observed in the dogs.
The ORF of the genes encoding TES-26, TES-32 and TES-120 proteins were cloned via RT-PCR method. This allow amplifications of cDNA copies of Toxocara mRNA, extracted from in vitro culture of L2 larvae, by using ‘gene’-specific downstream primers to prime cDNA synthesis. The TES-26, TES-32 and TES-120 gene fragments were then subcloned into PCR2.1 TOPO vector. The TOPO/TES-32 and TOPO/TES-120 DNA sequences analysis revealed that no amplification error (100% identity to their reported sequences in the Genbank) was generated during the gene amplification process. However, the TOPO/TES-26 gene fragment had four mutations with respect to their respective reported sequences in the Genbank which resulted in changes of amino acids of the protein.
These changes might be due to replication error occurred during PCR synthesis. However, all the base-errors in TOPO/TES-26 clone were successfully corrected by in-vitro PCR- based site directed mutagenesis.
The ORF of the genes encoding TES-26 and TES-32 were subsequently cloned into pET-42 vector version “b” and “a”, respectively, while TES-120 was cloned into pPROExTMHT version “a”, both vectors have a strong IPTG inducible promoter namely
T7lac promoter. After the cloning has been successfully performed, studies were conducted to determine the effect of different growth conditions on expression levels and to determine the solubility of the target proteins. For protein expression of the three recombinant constructs, IPTG at 1 mM was employed. High concentrations of IPTG (more than 1 mM
257 final concentration) led to an increased amount of insoluble protein. Over-expression of recombinant proteins up to 40% of total cell protein in E. coli often result in insoluble aggregates as inclusion bodies (Cabrita et al., 2004). The temperature was reduced to 30ºC after IPTG induction in order to decrease protein synthesis rate, hence promoting greater formation of soluble protein. Pilot studies on the proteins expressions revealed that 3, 4 and
5 hours of post-induction at 30°C with 1 mM IPTG was optimal for expression of soluble rTES-26, rTES-32 and rTES-120 recombinant proteins, respectively.
Since the recombinant proteins were produced in soluble form, purification under native condition was employed. IMAC technique using FPLC automated system was applied for purification of rTES-120 histidine-tagged protein using precharged Ni SepharoseTM High
Performance which offered significant times saving, high effectiveness and the target protein can be purified from a large volume of cell lysate in simple steps (Hochuli et al., 1988). A gradient washing of imidazole concentration in wash buffers was performed in the purification of histidine-tagged rTES-120 protein for the removal of undesirable impurities and to increase the protein purity. In contrast to the previously reported insoluble rTES-120 produced from bacterial expression host (Fong et al., 2003), the recombinant antigen produced in this study was soluble.
Fusion of the target protein with a highly soluble protein such as GST has been shown to increase the soluble fraction of the target protein (Smith & Johnson, 1988).
Laboratory-scale protein purification of rTES-26 and rTES-32 GST fusion proteins were carried out using a batch column controlled by gravity flow and not performed using FPLC.
This was because the automated machine requires a large volume of washing and elution buffers. In the present study, a total of 2.0, 1.5 and 1.5 mg of purified rTES-26, rTES-32 and rTES-120, respectively were obtained from 1 L of cell culture. The site-specific protease,
Factor Xa, was used to remove GST tag in the rTES-26 and rTES-32 GST-tagged fusion proteins. This study is the first report describing the use of GST tag in the expression and purification of Toxocara recombinant protein.
258 The rTES-26, rTES-32 and rTES-120 recombinant proteins were successfully purified to near homogeneity with high protein concentrations. The molecular weights of rTES-26, rTES-32 and rTES-120 recombinant proteins predicted using Vector NTi were 72,
64 and 31 kDa, respectively. These values corresponded with the size of the proteins observed using SDS-PAGE and Western blot. The Western blot analysis of rTES-26, rTES-
32 and rTES-120 revealed that the purified and/or cleaved recombinant antigens strongly reacted only with sera from patients infected with Toxocara tested against IgG4 antibodies.
These recombinant antigens showed no cross-reactivity with sera from healthy normals and sera from patients infected with STH.
Currently, diagnosis of human toxocariasis by commercial kits is performed by detecting total IgG antibody in the patient’s sera. These total IgG-assays lacked sufficient specificity since they gave many false-positive reactions with other parasitic helminth infections that are commonly found in developing tropical countries. These commercial IgG-
ELISAs using TES antigen are useful only for excluding toxocariasis from a differential diagnosis. However, the test interpretation can be problematic in cases of positive results
(Rahmah et al., 2005).
In this study, various antibody classes (IgE and IgM) and IgG subclasses (IgG1-4) were tested with the three recombinant antigens to find the best antibody that can produce the most sensitive and specific assay for diagnosis of toxocariasis. Checkerboard optimizations of ELISA were performed to determine the best concentrations of antigens, dilution of patients’ serum and secondary antibodies that can provide the highest discrimination between positive and negative samples.
The results clearly showed that only IgG4 assay displayed good specificity. The diagnostic utility of each purified recombinant antigens and rTES-30USM (previously produced in our laboratory) were further evaluated by IgG4-ELISA assay using 242 serum samples which included 30 sera from patients with clinical, haematological and serological evidence of toxocariasis. Both rTES-26 and rTES-32 IgG4-ELISAs demonstrated sensitivity of 80.0%; while rTES-120 and rTES-30USM IgG4-ELISAs showed sensitivity of 93.3.0%.
259 There was a concordance between the results in ELISA and Western blot results performed earlier i.e. positive or negative sera on the IgG4-ELISA assay yielded the same results as in
Western blot.
The sensitivity of rTES-120/rTES-30USM IgG4-ELISA was found to be significantly higher than rTES-26/rTES-32 IgG4-ELISA (p<0.001). However, the mean
O.Ds of the 30 toxocariasis samples among the IgG4 assays using the four recombinant antigens were shown not to be significantly different (p>0.05). This study is the first to demonstrate the use of rTES-120 with IgG4 assay. The rTES-26, rTES-32, rTES-120 and rTES-30USM gave specificities between 92.0 to 96.2%, with negative results observed when tested with all healthy individuals (N=100). The absence of significant cross-reactive sera from other related helminthic infections confirms the high specificity of the rTES-26, rTES-
32 and rTES-120 antigens in detecting anti-toxocara antibodies and its usefulness in a tropical region like Malaysia, where multiple helminthic infections especially STH are common. Most of the small number of cross-reactions were caused by STH (presumably false-positives), and this was not surprising as the ascarid nematodes are taxonomically related. Since environmental contamination with Toxocara and STH are common in the tropics, the patients infected with Toxocara species are likely to be infected with Ascaris
(Jones et al., 2008). Thus in this study, the possibility of Toxocara co-infections in the non- toxocara which shows false positive cannot be excluded. At p<0.05, there was marginally no significant difference between the specificities of rTES-26 and rTES-120 (p=0.059), between rTES-26 and rTES-30USM or between rTES-30USM and rTES-120.
The rTES-32 was excluded in the final development of the assay since it was not better than rTES-26 in terms of sensitivity or specificity. Instead rTES-30USM was included due to its high sensitivity and the fact that a 100% detection of toxocariasis cases was achieved when results of IgG4 assays using rTES-30USM and rTES-120 IgG4 were combined. A final assay which is sensitive (80.0% to 93.3%) and specific (92.0% to 96.2%) for detection of toxocariasis was successfully developed using three adjacent wells, each separately coated with rTES-26, rTES-30USM and rTES-120. The PPV of 64%-79%
260 suggest good reliability and accuracy of the tests developed, while the high NPV (95-99%) showed that the tests are capable of discriminating non-toxocara cases. When all assays are positive and the clinical symptoms are consistent with Toxocara infection, there is strong evidence/high likelihood that it is a true positive case. The same situations are applied when rTES-26 and rTES-120 ELISA or rTES-26 and rTES-30USM ELISA are positive. When only rTES-30USM or rTES-120 ELISA is positive, there is good evidence that the patient was infected.
In conclusion, the recombinant T. canis second-stage larva antigens corresponding to the 26, 32 and 120-kDa proteins of the TES secreted by infective larvae were successfully produced. The present study clearly demonstrated that these recombinant antigens are potentially good diagnostic reagents for serodiagnosis of Toxocara infection when used in
IgG4 assays. The rTES-30USM previously produced by our team was included in the final assay (and rTES-32 excluded) due to its high sensitivity and the observation that the combination of the results of rTES-30USM and rTES-120 produced 100% sensitivity. The use of multiple recombinant antigens would be expected to provide a more robust test as compared to the use of a single recombinant antigen, and this is the first report of such approach for diagnosis of toxocariasis. Thus, a highly sensitive and specific IgG4 assay for detection of toxocariasis was successfully developed using three adjacent wells, each separately coated with rTES-26, rTES-30USM and rTES-120. This would be very useful for routine serodiagnosis of toxocariasis worldwide, and would be especially beneficial for use in tropical countries where helminth infections are endemic.
Recommendation for Future Research
In this study, an ELISA assays using three recombinant antigens for detection of toxocariasis was developed based on detection of specific IgG4 antibodies in serum samples. Further multicentre studies need to be performed with a larger set of patients with Toxocara infection and other related infections to further confirm its usefulness in various settings. The
261 ELISA assays can also be upgraded to a simple assay device that can be completed within 15 minutes. Since, the current global diagnostic trend is moving towards the rapid platform, there is a need to develop the next generation of immunoassay technologies which provide a more rapid assay. Thus, a rapid, simple, highly sensitive and specific diagnostic test is highly desired for the diagnosis of human toxocariasis. One such technology is the immunochromatography platform which is based on membrane-based antibody assays and has been shown to be a potential tool for the diagnosis of infectious avgents (Weil et al.,
2000; Rahmah et al., 2001; Lammie et al., 2004). The principal advantages of the dipstick test are its rapidity, simplicity and the test can be done at site. Besides that, the dipstick is useful for use in health centres without adequate laboratory facilities.
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APPENDICES Appendix A LIST OF CHEMICALS AND REAGENTS
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) Sigma Aldrich, USA Absolute ethanol BDH Chemicals, UK Amido Black (Naphtol Blue Black) BDH Chemicals, UK Ammonium persulfate Amresco, USA Ampicillin Amresco, USA Bacteriological agar BDH Chemicals, UK Bacto-tryptone BDH Chemicals, UK Boric acid BDH Chemicals, UK Calcium chloride (CaCl2) Sigma Aldrich, USA Coomassie Brilliant Blue R250 BDH Chemicals, UK Diethyl pyrocarbonate (DEPC) Sigma Aldrich, USA Dipotassium hydrogen phosphate (K2HPO4) BDH Chemicals, UK
274 Disodium hydrogen phosphate (Na2HPO4) BDH Chemicals, UK DNAse 1 Amresco, USA EDTA (disodium salt dehydrate) Amresco, USA Ethidium bromide Sigma Aldrich, USA Fungizone Novagen, USA Glacial acetic acid BDH Chemicals, UK Glycerol Amresco, USA Glycine Amresco, USA Hydrochloric acid (HCl) Sigma Aldrich, USA Imidazole Amresco, USA Isopropanol Sigma Aldrich, USA Isopropyl-β-D- galactopyranoside (IPTG), Amresco, USA Kanamycin Amresco, USA Lysozyme Amresco, USA Magnesium chloride (MgCl2) Sigma Aldrich, USA Methanol BDH Chemicals, UK NaH2PO4 BDH Chemicals, UK NaHCO3 BDH Chemicals, UK Orange-G Sigma Aldrich, USA Penicillin Novagen, USA Pepsin Sigma Aldrich, USA Ponceau S Sigma Aldrich, USA Potassium chloride (KCl) BDH Chemicals, UK Potassium dihydrogen phosphate (KH2PO4) BDH Chemicals, UK Protease inhibitor cocktail Roche Diag, Germany SDS BDH Chemicals, UK Sodium carbonate (Na2CO3) BDH Chemicals, UK Sodium chloride (NaCl) Amresco, USA Sodium hydroxide (NaOH) BDH Chemicals, UK Sodium hypochlorite Sigma Aldrich, USA Streptomycin Novagen, USA Sucrose Sigma Aldrich, USA Tris base Amresco, USA Tris-HCl Amresco, USA Triton-X100 Sigma Aldrich, USA Tween 20 Amresco, USA Yeast extract BDH Chemicals, UK
Appendix B
LOCUS TCU29761 1034 bp mRNA linear INV 04-AUG-1995 DEFINITION Toxocara canis excretory-secretory antigen-26 (tes-26) mRNA, complete cds. ACCESSION U29761 VERSION U29761.1 GI:881974 SOURCE Toxocara canis (dog roundworm) ORGANISM Toxocara canis Eukaryota; Metazoa; Nematoda; Chromadorea; Ascaridida; Ascaridoidea; Toxocaridae; Toxocara. REFERENCE 1 (bases 1 to 1034) AUTHORS Gems,D., Ferguson,C.J., Robertson,B.D., Nieves,R., Page,A.P., Blaxter,M.L. and Maizels,R.M. TITLE An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins
275 JOURNAL J. Biol. Chem. 270 (31), 18517-18522 (1995) PUBMED 7629180 REFERENCE 2 (bases 1 to 1034) AUTHORS Maizels,R.M. TITLE Direct Submission JOURNAL Submitted (22-JUN-1995) Richard M. Maizels, Institute of Cell,Animal and Population Biology, Ashworth Laboratories, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, U.K FEATURES Location/Qualifiers source 1..1034 /organism="Toxocara canis" /mol_type="mRNA" /db_xref="taxon:6265" /dev_stage="infective larva" gene 1..1034 /gene="tes-26" CDS 10..798 /gene="tes-26" /function="phosphatidylethanolamine-binding protein" /standard_name="toxocara excretory-secretory antigen-26" /note="26 kDa protein secreted by the larval parasite" /codon_start=1 /product="TES-26" /protein_id="AAC46843.1" /db_xref="GI:881975" /translation="MSVVHKACLIALLFVSSGVAQQCMDSASDCAANAGSCFT RPVSQVLQNRCQRTCNTCDCRDEANNCAASINLCQNPTFEPLVRDRCQKTCGLCAG CGFISSGIVPLVVTSAPSRRVSVTFANNVQVNCGNTLTTAQVANQPTVTWEAQPND RYTLIMVD DFPSAANGQQGQRLHWWVINIPGNNIAGGTTLAAFQPSTPAANTGVHRYVFLVYRQ PAAINSPLLNNLVVQDSERPGFGTTAFATQFNLGSPYAGNFYRSQA"
polyA_site 1034 /gene="tes-26" /note="25 A nucleotides"
ORIGIN 1 ggtttcatca tgtcagttgt acacaaagct tgcttaatag ctttactgtt tgtgtcgagt 61 ggagtggcac aacagtgtat ggacagtgcg tcagactgtg cggcaaacgc tgggtcctgt 121 ttcacacgac cagtcagcca agttctgcag aacaggtgtc aaaggacatg caatacatgc 181 gactgccggg acgaggctaa caattgtgca gcttcaatta acctctgcca gaaccccaca 241 tttgaacctt tagttcgtga tcgatgccaa aagacttgtg gtttgtgtgc ggggtgcgga 301 ttcatcagta gcggaatcgt tccattagtt gtgacgagtg caccttcaag acgcgtgagt 361 gtgaccttcg ctaacaacgt tcaggtgaac tgcggtaata ctttgacgac ggcgcaagta 421 gccaaccaac cgacagtaac gtgggaagcg caaccgaatg acaggtacac cctcatcatg 481 gtcgatcctg acttcccaag cgcggcgaac ggccagcagg gtcagcgact tcactggtgg 541 gtaataaaca ttccaggaaa taatatcgcc ggtggtacga cgctcgcagc gttccagcca 601 tcaacacccg ccgctaatac tggtgtgcac cgatacgtgt ttctggtata cagacagcct 661 gccgcaataa actctccgct acttaataat ttggtagtgc aggattcgga gcggccaggt 721 tttggtacga ctgcctttgc cactcagttt aaccttggat caccctatgc tggcaatttc 781 tatcgatcgc aggcctaata gtcggtactt ccgatgtgca aataactccg cgagaaacta 841 gtacaaccac aactgctgtg aatactggac tccataatga tttagacctg atttccagaa
276 901 ttccagtttc ctcgaaattc tattgttttc ccgaagtccg tagattgcac aaccgtttct 961 gttttctttg cattgcagat gcctcccaga cttggagttc ttatcaattt gctaattaat 1021 aaacaagttg ttga
LOCUS AF041023 762 bp mRNA linear INV 02-SEP-1999 DEFINITION Toxocara canis excretory/secretory C-type lectin TES-32 (tes-32) mRNA, complete cds. ACCESSION AF041023 VERSION AF041023.1 GI:2773354 SOURCE Toxocara canis (dog roundworm) ORGANISM Toxocara canis Eukaryota; Metazoa; Nematoda; Chromadorea; Ascaridida; Ascaridoidea; Toxocaridae; Toxocara. REFERENCE 1 (bases 1 to 762) AUTHORS Loukas,A., Mullin,N.P., Tetteh,K.K., Moens,L. and Maizels,R.M. TITLE A novel C-type lectin secreted by a tissue-dwelling parasitic nematode JOURNAL Curr. Biol. 9 (15), 825-828 (1999) PUBMED 10469567 REFERENCE 2 (bases 1 to 762) JOURNAL Submitted (05-JAN-1998) ICAPB, University of Edinburgh, West Mains
277 Rd, Edinburgh EH9 3JT, United Kingdom FEATURES Location/Qualifiers source 1..762 /organism="Toxocara canis" /mol_type="mRNA" /db_xref="taxon:6265" /dev_stage="infective larva (L2)" gene 1..762 /gene="tes-32" CDS 26..685 /gene="tes-32" /note="similar to brevican and other proteins with C-type lectin domains" /codon_start=1 /product="excretory/secretory C-type lectin TES-32" /protein_id="AAB96779.1" /db_xref="GI:2773355" /translation="MIAAIVVLLCLHLSTTNACATNNDCGIFQVCVNNVCVANNQGCNPPCV APQVCVAPMCVAPPPAATTTAAPGVTTTRPRACPPNWTPFNNNCYIASLPGRFLQAS DWCTQTGSRVVWFDQSTVGNFGSELNFVNSFAVGRGVTRYWIGVNRQFGQWVFN GSPVIFSNWRPSQPDGCCGSNVTCAFVNYANFLGQWDDAPCGSLFTTPQGFVCKRP L“ misc_feature 266..670 /gene="tes-32" /note="C-type lectin domain; other site" ORIGIN 1 gcacgagcca acaaccacac gcaggatgat cgccgcaatc gtcgttttgt tatgtctcca 61 cctgtccacc acaaatgctt gcgccaccaa caatgactgt ggtattttcc aagtgtgcgt 121 taacaacgtc tgcgttgcca ataaccaagg atgcaatccg ccttgtgtgg ctccgcaggt 181 ttgcgttgca ccaatgtgtg tcgcccctcc accggcagcc actacaactg cagctccagg 241 cgttacaact acaagaccac gtgcctgccc acccaattgg acgccgttca acaacaattg 301 ctatatcgcc tctctacctg gacgcttcct tttcaaccaa gctagcgact ggtgtacaca 361 gactggctca agagttgtgt ggttcgacca gtcgactgta ggaaacttcg gcagcgaact 421 gaacttcgtc aacagtttcg ctgttggtcg tggagtaacc agatactgga taggagtgaa 481 cagacagttc ggccagtggg tgtttacgaa tggaagtcca gtgatatttt cgaactggag 541 gcctagtcaa ccggacggat gctgtggttc caacgtcacg tgtgcgttcg ttaattacgc 601 gaacttcctc ggccaatggg atgatgcccc atgcggaagt ttgtttacga ctccacaagg 661 cttcgtatgc aagagacctc tctagattca atgcctgctg cgctgatggc gtcttcgttt 721 cctgctgtgg taataaaatt cagacgaaaa aaaaaaaaaa aa LOCUS TCU39815 750 bp mRNA linear INV 08-AUG-1996 DEFINITION Toxocara canis surface coat glycoprotein TES-120 precursor (nmuc-1) mRNA, complete cds. ACCESSION U39815 VERSION U39815.1 GI:1103868 SOURCE Toxocara canis (dog roundworm) ORGANISM Toxocara canis Eukaryota; Metazoa; Nematoda; Chromadorea; Ascaridida; Ascaridoidea; Toxocaridae; Toxocara. REFERENCE 1 (bases 1 to 750) AUTHORS Gems,D. and Maizels,R.M. TITLE An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins JOURNAL Proc. Natl. Acad. Sci. U.S.A. 93 (4), 1665-1670 (1996) PUBMED 8643687 JOURNAL Submitted (01-NOV-1995) Rick Maizels, Institute of Cell, Animal and
278 Population Biology, Edinburgh University, King's Buildings, West Mains Road, Edinburgh, EH9 3JT, UK FEATURES Location/Qualifiers source 1..750 /organism="Toxocara canis" /mol_type="mRNA" /db_xref="taxon:6265" gene 1..750 /gene="nmuc-1" CDS 33..563 /gene="nmuc-1" /note="mucin-like apoprotein" /codon_start=1 /product="surface coat glycoprotein TES-120 precursor" /protein_id="AAB05820.1" /db_xref="GI:1103869" /translation="MHVLTVALVAVLICVATPQMMSSSSSSSPSTSSSSASTSSSS AS TSSSSASTSSSSASTSSSPASTSSSSASTSSMAGSTSTAAGPTSSSSVSTSTPAVMTT TPACIDTANDCQLFTPLCFVQPYSRAIQGRCRRTCNICSCQDSANDCANFVSVCLNPT YQPVLRSRCPLTCGFC" polyA_site 750
ORIGIN 1 ggttcaatta cccaagttgg aggagccacg caatgcacgt ccttaccgtc gctctcgtcg 61 cagtgctaat ctgcgttgct acaccacaaa tgatgtccag ttcctcttca tcgtctccgt 121 caacatcctc atcgtcagcg tcaacatcct catcgtccgc gtcaacatcc tcatcgtccg 181 cgtcaacatc ctcatcgtcc gcgtcaacat cctcatcgcc cgcgtcaaca tcctcatcgt 241 ccgcgtcaac aagctcaatg gcaggatcta catccacagc agctgggccg acatccagct 301 cttctgtttc cacatcaact ccagcggtga tgacaacgac gccagcgtgc atcgacaccg 361 ctaacgactg ccagctgttc acgccactct gcttcgttca gccatacagc agagcaatac 421 agggaagatg ccgaaggacg tgcaacattt gcagctgtca ggacagtgcc aacgactgtg 481 caaactttgt ttcagtctgc ctgaacccga cctatcagcc agtgcttcga tcaagatgcc 541 cactgacgtg cggcttctgt taatgatcgg tagtatcgcg acgaactgaa tggctttctc 601 gaaactgtgg atatcattcg aaaagaacgc gcgtcaactt tgttgagctg actgcacgaa 661 aatgtgatgg gaaaatacgt ttactctatt tttgaatgaa gtattcttaa tctccctctt 721 tgttttcgat aaaggagttc atgcaagaga
LIST OF PUBLICATIONS, PRESENTATIONS AND PATENT
PUBLICATION 1
Development and evaluation of a sensitive and specific assay for human toxocariasis using three recombinant antigens (TES-26, TES-30, and TES-120)
Journal of Clinical Microbiology. 2009 Apr 15
Suharni Mohamad, Norhaida Che Azmi and Rahmah Noordin
Abstract
Presently the laboratory diagnosis for of human toxocariasis currently relies on serological serologic tests which employ that use Toxocara excretory-secretory (TES) antigen in detecting to detect immunoglobulin (Ig)G antibodies to the larvae. In general, however, these
279 assays do not have good adequate specificity for use in countries endemic with in which other soil-transmitted helminthes are endemic. The use of recombinant antigens in these assays, however, is promising for improved improving the specificity of the diagnosis of toxocariasis. Towards this goal, we developed an IgG4-enzyme-linked immunosorbent assay (ELISA) assay which employing three recombinant antigens: r-TES30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-polymerase chain reaction (RT-PCR) cloning, subcloned into a glutathione S- transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively, and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested using various immunoglobulin classes (IgG, IgM, IgE) and IgG subclasses. The IgG4-ELISA assay was found to be most specific and have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4-ELISA showed 80.0% (24/30) sensitivity of 80.0% (24/30), while and both the rTES- 30USM IgG4-ELISA and rTES-120 IgG4-ELISA showed sensitivity of 93.3.0% (28/30). Combined use of rTES-120- and TES-30 IgG4-ELISA for the diagnosis of toxocariasis, provided 100% sensitivity. There was significant difference between the specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. Our results showed that the development of a diagnostic test which used three recombinant antigens will enable the development of a robust test and allow for more accurate detection of toxocariasis.
PUBLICATION 2 rTES-30USM: cloning via assembly PCR, expression, and evaluation of usefulness in the detection of toxocariasis.
Ann Trop Med Parasitol. 2008 Mar; 102(2):151-60 Norhaida A, Suharni M, Liza Sharmini AT, Tuda J, Rahmah N.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
Currently, the laboratory diagnosis of toxocariasis, caused by Toxocara canis or T. cati, mainly relies on serological tests. Unfortunately, however, the specificities of most of the commercial tests that are available for the serodiagnosis of this disease are not very high and this may cause problems, especially in tropical countries where co-infections with other helminths are common. In an effort to develop a serological assay with improved specificity for the detection of Toxocara infection, an IgG(4)-ELISA based on a recombinant version (rTES-30USM) of the 30-kDa Toxocara excretory-secretory antigen (TES-30) has recently been developed. To produce the antigen, the TES-30 gene was cloned via assembly PCR, subcloned into a His-tagged prokaryotic expression vector, and purified by affinity chromatography using Ni(2+)-nitrilotriacetic-acid (Ni-NTA) resin. The performance of the
280 ELISA based on the recombinant antigen was then compared with that of commercial kit, based on an IgG-ELISA, for the serodiagnosis of toxocariasis (Toxocara IgG-ELISA; Cypress Diagnostics, Langdorp, Belgium). Both assays were used to test 338 serum samples, including 26 samples from probable cases of toxocariasis. Assuming that all the probable cases were true cases, the assay based on rTES-30USM demonstrated a sensitivity of 92.3% (24/26) and a specificity of 89.6% (103/115) whereas the commercial kit exhibited a sensitivity of 100% (26/26) but a specificity of only 55.7% (64/115). The high sensitivity and specificity exhibited by the new IgG(4)-ELISA should make the assay a good choice for use in tropical countries and any other area where potentially cross-reactive helminthic infections are common.
PUBLICATION 3
Comparison of IgG-ELISA and IgG4-ELISA for Toxocara serodiagnosis. Acta Trop. 2005 Jan; 93(1):57-62
Rahmah N, Smith HV, Suharni M, Maizels RM, Fong MY
Institute for Research in Molecular Medicine, Suite 110, Eureka Complex, Universiti Sains Malaysia, 11800 Penang, Malaysia. [email protected]
Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4
281 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.
PUBLICATION 4
The use of dual recombinant antigens for improved sensitivity in diagnosis of human toxocariasis
The Malaysian Journal of Medical Sciences, Volume 14 Supplement 1, 2007, pg 42.
Suharni M, Norhaida A, Liza Sharmini AT & Rahmah N.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
Introduction: Current laboratory diagnosis for toxocariasis relies heavily on TES (toxocara excretory-secretory)-based serological tests. Since TES production is limited due to the capacity of parasite culture, the use of recombinant antigens which can be produced in unlimited amounts, offers a good alternative for diagnosis for toxocariasis. In striving towards this goal, we have developed an IgG4-ELISA which employs TES-30 (rTES-30) and TES-120 (rTES-120) recombinant antigens. Both these recombinant antigens have been previously reported to be of potential diagnostic value.
282 Methodology: The TES-30 gene was previously cloned; while the TES-120 gene was cloned via RT-PCR, subcloned into a His-tagged prokaryotic expression vector and expressed in E. coli. The recombinant proteins were subsequently purified by affinity chromatography using Ni-NTA resin. The diagnostic utility of each purified recombinant antigen (rTES30 and rTES120) and a combination of these two recombinant antigens were evaluated by IgG4-ELISA using 28 serum samples from patients infected with Toxocara.
Results: rTES-30 ELISA and rTES-120 ELISA showed sensitivity of 92.3% and 92.8% respectively for diagnosis of toxocariasis. In 9 out of 28 (32%) serum samples, the optical densities shown by the two recombinant antigens were significantly different. However when the results of both recombinant antigens were employed in the analysis of the sensitivity of the assay, all 28 samples displayed positive results (100% sensitive). Thus, consideration of the results of both recombinant antigens was shown to improve the diagnosis of toxocariasis.
Conclusion: In conclusion, our results demonstrated that a diagnostic test which employs dual recombinant antigens namely rTES-30 and rTES-120, allows for the establishment of a more sensitive and robust test as compared to a test which is based on single recombinant antigen.
PRESENTATION 1
The use of dual recombinant antigens for improved sensitivity in diagnosis of human toxocariasis
Presented as oral presentation at National Conference on Medical Sciences, Health Campus, USM Kubang Kerian, Kelantan. 18-21 May 2007.
Suharni M, Norhaida A, Liza Sharmini AT & Rahmah N.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
Introduction: Current laboratory diagnosis for toxocariasis relies heavily on TES (toxocara excretory-secretory)-based serological tests. Since TES production is limited due to the capacity of parasite culture, the use of recombinant antigens which can be produced in unlimited amounts, offers a good alternative for diagnosis for toxocariasis. In striving towards this goal, we have developed an IgG4-ELISA which employs TES-30 (rTES-30)
283 and TES-120 (rTES-120) recombinant antigens. Both these recombinant antigens have been previously reported to be of potential diagnostic value.
Methodology: The TES-30 gene was previously cloned; while the TES-120 gene was cloned via RT-PCR, subcloned into a His-tagged prokaryotic expression vector and expressed in E. coli. The recombinant proteins were subsequently purified by affinity chromatography using Ni-NTA resin. The diagnostic utility of each purified recombinant antigen (rTES30 and rTES120) and a combination of these two recombinant antigens were evaluated by IgG4-ELISA using 28 serum samples from patients infected with Toxocara.
Results: rTES30-ELISA and rTES-120-ELISA showed sensitivity of 92.3% and 92.8% respectively for diagnosis of toxocariasis. In 9 out of 28 (32%) serum samples, the optical densities shown by the two recombinant antigens were significantly different. However when the results of both recombinant antigens were employed in the analysis of the sensitivity of the assay, all 28 samples displayed positive results (100% sensitive). Thus, consideration of the results of both recombinant antigens was shown to improve the diagnosis of toxocariasis.
Conclusion: In conclusion, our results demonstrated that a diagnostic test which employs dual recombinant antigens namely rTES-30 and rTES-120, allows for the establishment of a more sensitive and robust test as compared to a test which is based on single recombinant antigen.
284 PRESENTATION 2
Development of a sensitive and specific recombinant antigen-based assay for diagnosis of human toxocariasis
Accepted as oral presentation at National Conference on Environmental Health, Grand Riverview Hotel, Kelantan. 29-30 October 2008.
Suharni, M and Rahmah N
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Penang, Malaysia
Presently serodiagnosis of human toxocariasis are based on indirect IgG-ELISA kits which employ native Toxocara excretory-secretory (TES) antigens. However, these assays lacked specificity especially for use in tropical countries where multiparasitism are prevalent. In an effort to improve the diagnostic test for this infection, we have developed an IgG4-ELISA assay which uses three recombinant antigens.
T. canis recombinant TES-26, TES-32 and TES-120 antigens were produced in prokaryotic expression vectors and expressed in E. coli. The diagnostic utility of these antigens were evaluated using serum samples from 100 healthy individuals and 141 patients with various infections namely 30 toxocariasis, 28 soil-transmitted helminthes (STH), 30 amoebiasis, 20 toxoplasmosis, 28 brugian filariasis and 5 with strongyloidiasis.
Both rTES26- and rTES32-ELISA showed 80.0% sensitivities, while rTES120-ELISA showed sensitivity of 93.3.0%. The specificities of rTES26, rTES32 and rTES120 antigens were 96.2, 93.9 and 92.0%, respectively. Our results showed that a diagnostic test based on IgG4 assay using these highly specific recombinant antigens will provide a sensitive and specific assay for diagnosis of toxocariasis.
285 PATENT FILED
USM has filed a Malaysian patent and PCT patent entitled, “Recombinant antigen for detection of toxocariasis”.
286