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Application of Recombinant Antibody Technology for the Development of Anti-Lipid Antibodies for Tuberculosis Diagnosis

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Application of Recombinant Antibody Technology for the Development of Anti-Lipid Antibodies for Tuberculosis Diagnosis APPLICATION OF RECOMBINANT ANTIBODY TECHNOLOGY FOR THE DEVELOPMENT OF ANTI-LIPID ANTIBODIES FOR TUBERCULOSIS DIAGNOSIS CONRAD CHAN EN ZUO NATIONAL UNIVERSITY OF SINGAPORE 2013 APPLICATION OF RECOMBINANT ANTIBODY TECHNOLOGY FOR THE DEVELOPMENT OF ANTI-LIPID ANTIBODIES FOR TUBERCULOSIS DIAGNOSIS CONRAD CHAN EN ZUO BSc. (Hons.), MRes. Imperial College London A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously __________________________ Conrad Chan En Zuo 5th August 2013 Acknowledgements Acknowledgements The work here would not have been possible without the assistance of so many people. Firstly, to A/Prof Paul MacAry and Dr. Brendon Hanson, my co- supervisors, thank you for your encouragement, advice, support and the opportunity to carry out research in a very exciting field. Also to my collaborators with whom I had the privilege of working with over these five years; From NUS: Dr Timothy Barkham, Dr Seah Geok Teng, Prof Markus Wenk, Dr Anne Bendt, Dr Amaury Cazenave-Gassiot; From FIND: Dr Gerd Michel, From Max Planck Institute Berlin: Prof Peter Seeberger & Sebastian Gotze, From Georgia: Dr Nestan Tukvadze and the staff of the TB Institute, Dr Mason Soule and Dr Mzia Kutateladze, I really appreciate the sharing of your scientific expertise and efforts. A special note of thanks to those in Georgia, who made my trip a real pleasure. To all my fellow colleagues at DSO National Laboratories, Annie, Steve, Angeline, Shyue Wei, De Hoe, Grace and Shirley, thanks for all your assistance and encouragement and for covering all the stuff I could not do. The same to my fellow students & colleagues in PAM Lab especially those in the Lipid Squad: Omedul and Yanting, as well as Fatimah for doing all those admin stuff that we hate to do. I would also like to acknowledge the support of DSO National Laboratories for providing the scholarship to support my studies. Finally, to God, for His innumerable blessings and provision along the way; and to my family and especially my wife Sally, this is as much your success as it is mine. i Table of Contents Table of Contents Acknowledgements .................................................................................................... 1 Table of Contents ........................................................................................................ ii Summary .................................................................................................................. viii List of Tables .............................................................................................................. x List of Figures ............................................................................................................. xi List of Abbreviations ................................................................................................. xiv List of Publications & Patents .................................................................................. xvii Chapter 1: Introduction ............................................................................................... 1 1.1 Tuberculosis pathology, epidemiology, prevention and treatment .................... 2 1.2 Methods for TB diagnosis ................................................................................. 7 1.2.1 Detection of host immune responses- X-rays, TST and IGRA ................... 8 1.2.2 Direct microbiological detection – Acid-fast stain, microbial culture and Nucleic acid amplification tests ........................................................................... 9 1.3 TB diagnostics in resource-poor settings ........................................................ 12 1.3.1 Improving current diagnostics for resource-poor countries ...................... 13 1.3.2 Potential point-of care diagnostics for resource-poor countries................ 15 1.4 Anti-lipid antibodies ......................................................................................... 18 1.4.1 Lipids as disease biomarkers ................................................................... 18 1.4.2 Recombinant phage display ..................................................................... 20 1.4.3 Recombinant antibody expression ........................................................... 21 ii Table of Contents 1.5 Lipid biomarkers for TB diagnostics ................................................................ 23 1.5.1 Lipoarabinomannan ................................................................................. 25 1.5.2 LAM diagnostics ....................................................................................... 29 1.5.3 Mycolic acid .............................................................................................. 31 1.6 Aims of this thesis ........................................................................................... 35 1.6.1 Optimize expression of full length IgG in E. coli ....................................... 36 1.6.2 Develop antibodies targeting the Mtb lipids Lipoarabinomannan and mycolic acid ...................................................................................................... 36 1.6.3 Thoroughly characterize anti-LAM/anti-mycolic acid antibodies ............... 37 1.6.4 Determine the diagnostic utility of the antibodies ..................................... 37 Chapter 2: Materials and methods ........................................................................... 38 2.1 Buffers and solutions ...................................................................................... 39 2.2 Construction of antibody expression vectors .................................................. 42 2.2.1 Construction of mammalian expression vectors ....................................... 42 2.2.2 Construction of bacterial expression vectors ............................................ 43 2.2.3 Construction of chimeric antibody constructs ........................................... 43 2.2.4 Sub-cloning of antibody heavy and light chains by restriction digest and ligation ............................................................................................................... 46 2.3 Expression and purification of bacterial lgG .................................................... 46 2.3.1 Initial periplasmic expression in BL21 or HB2151 .................................... 46 2.3.2 Small scale optimization of bacterial IgG expression conditions .............. 47 2.3.3 Large-scale expression and purification of bacterial IgG .......................... 47 iii Table of Contents 2.4 Expression and purification of mammalian IgG and Fab ................................ 48 2.5 Polyacrylamide gels and western blot ............................................................. 50 2.6 Measurement of protein concentration ........................................................... 50 2.7 Bacterial strains and culture ........................................................................... 51 2.8 Phage display ................................................................................................. 52 2.8.1 Negative selection panning against ManLAM .......................................... 52 2.8.2 Lipid panning against mycolic acid ........................................................... 53 2.8.3 Phage recovery after each round of panning ........................................... 54 2.8.4 Screening of phage libraries ..................................................................... 55 2.9 Carbohydrate microarrays .............................................................................. 57 2.10 Immunofluorescence and acid fast-staining .................................................. 58 2.11 Collection and processing of bacterial cultures for ELISA ............................ 59 2.11.1 Bacterial supernatants and whole cell suspension for LAM ELISA ........ 59 2.11.2 Lipid extraction from bacterial cultures for mycolic acid ELISA .............. 60 2.12 Collection of clinical samples for ELISA ........................................................ 61 2.12.1 Spiked whole blood and serum samples for LAM ELISA ....................... 61 2.12.2 TB patient selection criteria and sample collection procedure ................ 62 2.12.3 Clinical patient serum samples for LAM ELISA ...................................... 63 2.12.4 Clinical patient urine samples for LAM ELISA ........................................ 63 2.12.5 Clinical patient sputum samples for LAM ELISA .................................... 63 2.12.6 Clinical patient sputum samples for mycolic acid ELISA ........................ 64 iv Table of Contents 2.13 ELISAs .......................................................................................................... 64 2.13.1 Comparison of functional IgG levels in bacterial lysate and determination of purified bacterial IgG affinity curves by indirect ELISA .................................. 64 2.13.2 Indirect phage polyclonal and monoclonal ELISAs ................................ 66 2.13.3 Indirect monoclonal IgG ELISA against LAM or lipids ............................ 67 2.13.4 Determination of chimeric antibody affinity binding curves ..................... 68 2.13.5 Determination
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