application of recombinant antibody technology for the development of anti-lipid antibodies for ...

240 Pages·2014·5.33 MB·English

by  Conrad Chan

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 of limit of sensitivity for anti-mycolic acid antibodies ....... 69
2.13.6 Indirect sandwich ELISA on purified LAM, bacterial suspensions and
culture supernatants .......................................................................................... 69
2.13.7 Determination of anti-LAM antibody titres in healthy serum samples ..... 70
2.13.8 Indirect sandwich ELISA on spiked or patient clinical samples .............. 71
2.13.9 Indirect ELISA on patient lipid extracts ................................................... 71
2.14 Mass spectrometric profiling and quantification of mycolic acids. ................. 72
2.15 Data analysis and statistics........................................................................... 73
Chapter 3: Optimization of IgG expression in bacteria ............................................. 74
3.1 Introduction ..................................................................................................... 75
3.2 Preliminary expression in two common E. coli bacterial strains ...................... 76
3.3 Optimization of expression in small scale culture ........................................... 78
3.4 Comparison of yield by large scale expression ............................................... 83
3.5 Comparison of bacterial and mammalian expressed IgG ............................... 85
3.6 Discussion ...................................................................................................... 87
Chapter 4: Generation of anti-ManLAM antibodies by phage display ...................... 91
v
Table of Contents

4.1 Introduction ..................................................................................................... 92
4.2 Panning of the Humanyx phage library ........................................................... 93
4.3 Monoclonal screening and identification of my2F12 ....................................... 95
4.4 Characterization of my2F12 specificity ........................................................... 97
4.5 Expression of my2F12 in bacteria ................................................................. 101
4.6 Discussion .................................................................................................... 105
Chapter 5: Optimization of my2F12 antibody and sample processing for diagnostic
use ......................................................................................................................... 108
5.1 Introduction ................................................................................................... 109
5.2 Design and expression of my2F12 chimeric antibodies ................................ 111
5.3 Characterization of my2F12 chimeric antibody avidity .................................. 114
5.4 Identification of pathogenic mycobacteria with chimeric my2F12 by
immunofluorescence microscopy ....................................................................... 115
5.5 Identification of pathogenic mycobacteria with chimeric my2F12 by sandwich
ELISA ................................................................................................................. 120
5.6 Enhancing the sensitivity of my2F12 ELISA on spiked serum samples ........ 122
5.7 Discussion .................................................................................................... 127
Chapter 6: Generation of anti-mycolic acid antibodies by phage display ............... 131
6.1 Introduction ................................................................................................... 132
6.2 Isolation of mycolic acid-specific antibodies ................................................. 133
6.3 Characterization of mycolic acid antibody specificity and sensitivity ............. 137
6.4 Optimization of mycolic acid extraction protocol ........................................... 142
6.5 Determination of species specificity .............................................................. 146
vi