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Production and Characterization of Monoclonal Antibody Against Campylobacter Species, Production and Characterization of Monoclo

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Production and Characterization of Monoclonal Antibody Against Campylobacter Species, Production and Characterization of Monoclo PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST CAMPYLOBACTER SPECIES BY MRS. NARISSARA MUNGKORNKAEW A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF MEDICAL TECHNOLOGY GRADUATE PROGRAM FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2016 COPYRIGHT OF THAMMASAT UNIVERSITY Ref. code: 25595412030115XYL PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST CAMPYLOBACTER SPECIES BY MRS. NARISSARA MUNGKORNKAEW A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF MEDICAL TECHNOLOGY GRADUATE PROGRAM FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2016 COPYRIGHT OF THAMMASAT UNIVERSITY Ref. code: 25595412030115XYL Ref. code: 25595412030115XYL Ref. code: 25595412030115XYL (1) Thesis Title PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST CAMPYLOBACTER SPECIES Author Mrs. Narissara Mungkornkaew Degree Master of Medical Technology Department/Faculty/University Faculty of Allied Health Sciences Thammasat University Master Advisor Associate Professor Worada Samosornsuk, D.V.Sc. Master Co-Advisors Professor Wanpen Chaicumpa, D.V.M. (Hons.), Ph.D. Assistant Professor Anek Pootong, Ph.D. Academic Year 2016 ABSTRACT Campylobacter species are an important cause of gastroenteritis in human. Immunological tools are needed for accurate and rapid identification of Campylobacter. We produced a monoclonal antibody (MAb) against Campylobacter by the fusion of P3X myeloma cells and spleen cells from BALB/c mice immunized with whole cell lysate of C. jejuni. The MAb, designated MAb2D10 and of the immunoglobulin G1 isotype, was produced at high titer. The specificity of MAb2D10 against the Campylobacter species was determined using indirect ELISA and dot blot ELISA. MAb2D10 reacted with C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus but did not react with C. fetus, C. hyointestinalis and non-Campylobacter species. Western blotting showed that MAb2D10 bound to proteins migrating at molecular masses ranging from 19–72 kDa. MAb2D10 may be useful for the development of simple and rapid diagnostic tools for detection of human Campylobacter infection. Keyword: Campylobacter; Monoclonal antibody; Gastroenteritis Ref. code: 25595412030115XYL (2) ACKNOWLEDGEMENTS I would like to express my sincere appreciation and gratitude to my advisor, Associate Professor Dr. Worada Samosornsuk, for her supervision, encouragement and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been invaluable. I am deeply grateful to my Co-advisor Professor Dr. Wanpen Chaicumpa and Assistant Professor Dr. Anek Pootong who gave useful suggestion and helpful discussion. I am also deeply grateful to my committee members, Associate Professor Dr. Nitat Sookrung, Assistant Professor Dr. Pongsri Tongtawe, and Assistant Professor Dr. Sekson Samosornsuk for their constructive comments and suggestions in this study. I express special thanks to Assistant Professor Dr. Srinuan Somroop for teaching and helping me every step. Without her guidance and constant feedback this thesis would not have been achievable. I wish to thank laboratory members of Medical Technology Department, Faculty of Allied Health Sciences, Ms. Nattharee Thanawan, Mr. Sompoch Prachan, Ms. Benja Narapong, Ms. Mattika Phunhkrachuy for assisting in laboratory work. I would especially like to thank microbiology laboratory members of Thammasat University Hospital. All of you have been there to support me, work hard for me when I studied. Last but not least, I would like to express my deep gratitude to my family, especially my father who inspired me to intend the educations. I would also like to say a heartfelt to my family for always believing in me and encouraging me to follow my dreams. Mrs. Narissara Mungkornkaew Ref. code: 25595412030115XYL (3) TABLE OF CONTENTS Page ABSTRACT (1) ACKNOWLEDGEMENTS (2) LIST OF TABLE (8) LIST OF FIGURE (9) LIST OF ABBREVIATIONS (10) CHAPTER 1 INTRODUCTION 1 1.1 General introduction 1 1.2 Objective 3 CHAPTER 2 REVIEW OF LITERATURE 4 2.1 Campylobacter 4 2.1.1 History 4 2.1.2 Taxonomy 5 2.1.3 Morphology 8 2.1.4 Biochemical properties 10 2.2 Culturing of Campylobacter 11 2.3 Pathogenesis of Campylobacter 12 2.3.1 Motility 15 2.3.1.1 Flagella 15 2.3.1.2 Chemotaxis 15 2.3.2 Adhesion 18 2.3.3 Invasion 20 2.3.4 Toxin production 21 2.3.5 Carbohydrate structures 22 2.3.6 Iron uptake system 22 2.3.7 Multidrug and bile resistance 22 Ref. code: 25595412030115XYL (4) 2.3.8 Antimicrobial resistance 22 2.3.9 Stress response and survival 22 2.4 Epidemiology of Campylobacter 24 2.5 Antibiotic resistance and sensitivity 26 2.6 Identification and detection of Campylobacter 28 2.6.1 Isolation of Campylobacter 28 2.6.1.1 Selective media for isolation 28 2.6.1.2 Passive filtration 29 2.6.1.3 Incubation 29 2.6.2 Confirmation 29 2.6.2.1 Identification on solid medium 29 2.6.2.2 Microscopic examination of morphology 30 and motility 2.6.2.3 Detection of oxidase 30 2.6.2.4 Microaerobic growth at 25°C 30 2.6.2.5 Aerobic growth at 42°C 30 2.6.2.6 Latex agglutination tests 30 2.6.3 Identification of Campylobacter to the 30 species level 2.6.3.1 Detection of hippurate hydrolysis 30 2.6.3.2 Detection of indoxyl acetate hydrolysis 31 2.6.4 Molecular detection of Campylobacter 32 2.6.4.1 Detection and speciation of 32 thermophilic Campylobacter species by molecular techniques 2.6.4.2 Subtyping of Campylobacter spp. 32 2.6.4.3 Flagellin typing (fla typing) 33 2.6.4.4 Pulsed Field Gel Electrophoresis (PFGE) 33 2.6.4.5 Ribotyping 34 2.6.4.6 Random Amplyfied polymorphic DNA (RAPD) 34 2.6.4.7 Amplified Fragment Length 35 Polymorphism (AFLP) 2.6.5 Immunological methods for detection of 35 Campylobacter spp. 2.6.5.1 Identification of Campylobacter spp. 37 using immunoassays Ref. code: 25595412030115XYL (5) (1) Clinical stool samples 37 (2) Food samples 38 2.6.5.2 Increasing concentration of campylobacters 39 prior to immunoassay identification (1) Enrichment of the samples 39 (2) Use of filter membranes to separate 39 contaminating bacteria 2.7 Antibody 40 2.8 Monoclonal antibody 44 2.8.1 History 44 2.8.2 Monoclonal antibody production 44 2.8.2.1 Immunization schedule 45 2.8.2.2 Myeloma cell line culture 47 2.8.2.3 Fusion 48 2.8.2.4 Growth and selection of 48 monoclonal antibodies 2.8.2.5 Long-term maintenance and 49 cryopreservation of MAbs 2.8.3 Monoclonal antibody characterization 49 2.8.3.1 Physicochemical characterization 49 2.8.3.2 Immunological properties 50 2.8.3.3 Biological activity 50 2.8.3.4 Purity, impurity and contaminants 50 2.8.3.5 Quality 51 2.8.4 Monoclonal antibodies application 51 2.8.4.1 Diagnostic application 51 (1) MAbs in biochemical analysis 51 (2) MAbs in diagnostic imaging 52 2.8.4.2 Therapeutic application 52 (1) MAbs as direct therapeutic agents 52 (2) MAbs as targeting agents in therapy 52 2.8.4.3 Protein purification 53 2.8.4.4 Miscellaneous applications 53 (1) Catalytic MAbs (ABZYMES) 53 (2) Autoantibody fingerprinting 54 2.9 Monoclonal antibodies against Campylobacter spp. 54 Ref. code: 25595412030115XYL (6) CHAPTER 3 RESEARCH METHODOLOGY 56 3.1 Experimental animals and bacterial strains 56 3.1.1 Experimental animals 56 3.1.2 Bacterial strains 56 3.2 Antigens preparation 66 3.2.1 Bacteria culture and storage 66 3.2.2 Whole cell lysate preparation by 66 ultrasonication technique 3.2.2.1 Preparation of the cell suspension 66 3.2.2.2 Ultrasonic lysis 66 3.2.3 Dry weight measurement of whole cell lysate 67 3.2.3.1 Aluminum dishes preparation 67 3.2.3.2 Measurement 67 3.2.3.3 Calculation 67 3.3 Immunization 68 3.4 Hybridoma production 70 3.4.1 Splenectomy 70 3.4.2 Preparation of myeloma cells 70 3.4.3 Fusion 70 3.4.3.1 Feeder preparation 71 3.4.3.2 Hybridoma screening 72 3.5 Monoclonal antibody production 72 3.5.1 Selection of positive hybridomas 72 3.5.2 Limiting dilution 72 3.6 Storage and revival of hybridomas 73 3.7 Monoclonal antibody characterization 75 3.7.1 Isotyping immunoglobulin subclasses 75 3.7.2 Sodium Dodecyl Sulfate Polyacrylamide Gel 76 Electrophoresis (SDS-PAGE) 3.7.3 Western blot 78 3.7.4 Indirect ELISA 80 3.7.5 Dot blot ELISA 81 Ref. code: 25595412030115XYL (7) CHAPTER 4 RESULTS AND DISCUSSION 83 4.1 Monoclonal antibody production 83 4.2 Monoclonal antibody characterization 85 4.2.1 Isotyping immunoglobulin subclasses 85 4.2.2 SDS-PAGE and western blot profile 85 4.2.3 Cross reactivity of MAb 89 4.3 Discussion 90 CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 93 REFERENCE 94 APPENDIX 108 BIOGRAPHY 118 Ref. code: 25595412030115XYL (8) LIST OF TABLES Tables Page 2.1 A list of species, subspecies, and biovars within the 6 Campylobacter genus. 2.2 Campylobacter motility and chemotaxis factors. 17 2.3 Campylobacter adhesion factors. 18 2.4 Campylobacter invasion factors. 20 2.5 Other virulence factors in Campylobacter. 21 2.6 Multidrug and bile resistance and stress response 23 virulence factors in Campylobacter. 2.7 Confirmatory tests for thermophilic Campylobacter. 31 2.8 Basic phenotypic characteristics of selected thermophilic 31 Campylobacter species. 2.9 Characteristics of human immunoglobulin isotypes. 41 2.10 Monoclonal antibodies against Campylobacter spp. 55 3.1 Bacteria used in this study. 57 4.1 Indirect ELISA reactivity of hybridoma supernatant 83 against C. jejuni43. 4.2 Indirect ELISA reactivity of 2D10 reclone supernatant 84 against C.
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