THE PRODUCTION OF ANTIBODIES TO COUMARIN AND ITS MAJOR HUMAN METABOLITES A ...

350 Pages·2013·7.51 MB·English

THE PRODUCTION OF ANTIBODIES TO COUMARIN AND ITS
MAJOR HUMAN METABOLITES
A thesis submitted for the degree of Ph.D.
by
Anthony Killard B.A. (Mod).
July 1998
Based on research carried out at
School of Biological Sciences
Dublin City University
Dublin 9
Ireland
Under the supervision of
Prof. Richard O’Kennedy
To Mum, Dad, Jackie and Kieran.
"An expert is a man who has made all the mistakes which can be made in a very
narrow field."
Niels Henrik Bohr
"I think I must be an expert by now."
Anthony J. Killard
ii
I hereby certify that this material, which I now submit for assessment on the programme
of study leading to the award of Ph.D. is entirely my own work and has not been taken
from the work of others save and to the extent that such work has been cited and
acknowledged within the text of my own work.
iii
ACKNOWLEDGMENTS
Although the previous page states that this work is all my own, no Ph.D. would be
possible without the help and support of so many people; family, friends and colleagues.
Many thanks to Richard for his patience and constant positive attitude. Thanks to my
family for giving me all the support I needed to get through this. To all the lab gang, past
(especially Mary, Teresa, Rob and Declan), and present (Stephen, Paul, Gary, Ciaran,
Deirdre, Brian and John). You have all been an inspiration. Special thanks to Mike who
helped me out of a few difficult patches. Also to the technical staff for putting up with
my all too frequent visits for so long. Thank you everybody.
iv
CONTENTS
Publications and Presentations xvi
Abstract xviii
Abbreviations and symbols xix
1.0 INTRODUCTION 1
1.1 THE IMMUNE SYSTEM 1
1.1.1 The Lymphoid system 1
1.1.2 The humoral immune response 3
1.2 ANTIBODY STRUCTURE 8
1.3 ANTIBODY CHAIN GENE RECOMBINATION 11
1.4 MONOCLONAL ANTIBODY TECHNOLOGY 13
1.5 ANTIBODY ENGINEERING 15
1.6 COMBINATORIAL PHAGE DISPLAY LIBRARIES 21
1.6.1 The pComb3 System 24
1.6.2 The Nissim Library 30
1.6.3 Bacterial surface display 30
1.7 ANTIBODY CONJUGATES AND CHIMAERAS 33
1.8 HUMANISED ANTIBODIES 36
1.9 AFFINITY MATURATION 39
1.10 CATALYTIC ANTIBODIES 40
1.11 ENGINEERED ANTIBODIES IN THE TREATMENT OF DISEASE 44
1.12 INTRACELLULAR IMMUNISATION 45
1.13 BISPECIFIC ANTIBODIES 56
1.13.1 Bispecific antibody therapy in association with
chemotherapeutic drugs 50
1.14 COUMARIN 50
1.14.1 The metabolism and pharmacokinetics of coumarin in man 51
1.14.2 Pharmacological role of coumarin and its metabolites 55
1.14.3 The analysis of coumarin and its metabolites 57
1.14.3.1 Physical methods of coumarin analysis 57
v
1.14.3.2 lmmunoanalytical techniques for coumarin analysis 57
1.14.4 Engineered antibodies forc oumarin analysis 59
1.15 AIMS 61
2.0 MATERIALS AND METHODS 62
2.1 MATERIALS 63
2.2 EQUIPMENT 64
2.3 METHODS 66
2.3.1 General protocols 66
2.3.1.1 Culture media formulations 66
2.3.1.2 Standard molecular biology protocols 67
2.3.1.3 Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) 67
2.3.1.4 Coomassie blue staining 67
2.3.1.5 Silver staining 68
2.3.1.6 Western blotting 68
2.3.1.7 Dot blotting 68
2.3.1.8 Bicinchoninic acid (BCA) assay 69
2.3.1.9 Production of anti-VC SMI 3 antibody in rabbit 69
2.3.1.10 Production and purification of anti-c-myc antibody from
mycl-9E10 70
2.3.1.11 Standard enzyme immunoassay protocol using polyclonal
antibodies 70
2.3.1.11.1 Non-competitive enzyme immunoassay 70
2.3.1.11.2 Competitive enzyme immunoassay 71
2.3.1.12 Standard enzyme immunoassay protocol using single chain
Fv antibodies 71
2.3.1.12.1 Non-competitive enzyme immunoassay 71
2.3.1.12.2 Competitive enzyme immunoassay 72
2.3.1.13 Agarose gel electrophoresis 72
vi
2.3.2 Production of murine Fab antibody libraries to coumarin and 7-
bydroxycoumarin 73
2.3.2.1 Production of a coumarin-thyroglobuttn conjugate 73
2.3.2.2 Characterisation of the coumarin-thyroglobulin conjugate 74
2.32.3 Spectrophotometric estimation of drug-protein coupling ratios 74
2.3.2.4 Immunisation of mice with coumarin-thyroglobulin and 7-
hydroxycoumarin-thyroglobulin 74
2.3.2.5 Preparation of mouse spleen messenger RNA 75
2.3.2.6 Reverse transcription of mouse spleen mRNA 76
2.3.2.7 Amplification of antibody light and heavy chain genes using
polymerase chain reaction 77
2.3.2.7.1 PCR Primers 77
2.3.2.7.2 PCR reaction 78
2.3.2.7.3 Purification of PCR reaction products 79
2.3.2.8 Insertion of antibody heavy or light chain genes into the
pGEM-T plasmid vector 79
2.3.2.9 Preparation of electrocompetent Escherichia coli XLl-Blue cells 79
2.3.2.10 Measurement of transformation efficiency of
electrocompetent E. coli XLl-Blue cells 80
2.3.2.11 Transformation of E. coli XLl-Blue with pGEM-T plasmid
vector containing light and heavy chain gene inserts 80
2.3.2.12 Isolation of light and heavy chain gene inserts from pGEM-T 81
2.3.2.13 Preparation of pComb3 phagemid vector for insert ligation
of antibody heavy chain genes 81
2.3.2.14 Transformation of E. coli XLl-Blue with pComb3 plasmid
vector containing light and heavy chain gene inserts 82
2.3.3 Production of single chain Fv antibodies to coumarin and 7-
hydroxycoumarin 83
2.3.3.1 The Nissim library 83
2.3.3.2 Growth of the Nissim Library 83
2.3.3.3 Preparation of phagemid particles 83
vii
2.3.3.4 Affinity selection of scFv antibodies using the Nissim library 84
2.3.3.5 Affinity selection of antibodies using BlAcore™ 85
2.3.3.6 Screening of scFv antibodies by Phage ELISA 86
2.3.3.6.1 Preparation of clones for phage ELISA 86
2.3.3.6.2 Phage ELISA 86
2.3.3.7 Transfer of pHENl to HB2151 for screening of soluble scFv
antibody expression 87
2.3.3.8 Screening of scFv isolates for soluble antibody expression 87
2.3.3.9 Analysis of the cellular distribution ofscFv antibodies 88
2.3.3.10 Large scale scFv antibody production 88
2.3.3.11 Production ofscFv antibody from intracellular compartments 89
2.3.3.12 Large scale phage antibody production 89
2.3.3.13 BlAcore™ kinetic analysis of anti-coumarin scFv antibody
clones 89
2.3.4 The in vitro production of 7-hydroxycoumarin glucuronide 90
2.3.4.1 Preparation of porcine and bovine UDP-glucuronyl transferase 90
2.3.4.2 Initial analysis of the in vitro production of 7-hydroxy coumarin-
glucuronide by HPLC 91
2.3.4.3 Repeat analysis of the in vitro production of 7-hydroxy coumarin-
glucuronide by HPLC 92
2.3.4.4 Analysis of the in vitro production of 7-hydroxy coumarin-
glucuronide by capillary electrophoresis (CE) 94
2.3.5 Purification of 7-hydroxycoumarin-glucuronide 95
2.3.5.1 Chromatographic separation of 7-hydroxycoumarin and 7-
hydroxycoumarin-glucuronide 95
2.3.5.2 Chromatographic purification of 7-hydroxycoumarin-glucuronide
from crude enzymatic reaction mixtures 95
2.3.5.3 Chromatographic purification of 7-hydroxycoumarin-glucuronide
from coumarin-treated urine 95
viii
2.3.5.4 Development of a methodology for the extraction of 7-
hydroxycoumarin-glucuronide from aqueous solutions 96
2.3.5.5 Extraction of 7-hydroxycoumarin-glucuronide from enzymatic
reaction mixtures and coumarin-treated patient urine 96
2.3.5.6 Assessment of the thermal stability of 7-hydroxy coumarin-
glucuronide at 6(fC 96
2.3.5.7 Assessment of the stability of 7-hydroxy coumarin-glucuronide to
a range of different pH conditions 96
2.3.5.8 In vitro enzymatic production of 3-amino-7-hydroxycoumarin-
glucuronide 97
2.3.5.8.1 Preparation of 3-amino-7-hydroxycoumarin 97
2.3.5.8.2 In vitro enzymatic production of 3-amino-7-
hydroxy coumarin-glucuronide 97
2.3.5.9 Production of a 7-hydroxycoumarin-glucuronide-bovine serum
albumin conjugate 97
3.0 PRODUCTION OF MURINE Fab ANTIBODY LIBRARIES TO
COUMARIN AND 7-HYDROXYCOUMARIN USING THE pComb3
SYSTEM 99
3.1 INTRODUCTION 100
3.1.1 Antibody phage display systems 100
3.1.2 Source and quality of material for library construction 100
3.1.3 Production of hapten-protein conjugates 101
3.1.4 T/A cloning vectors 101
3.2 RESULTS 103
3.2.1 Preparation of coumarin and 7-hydroxycoumarin protein conjugates 103
3.2.2 Production of coumarin-thyroglobulin (coumarin-THG) conjugate 103
3.2.3 Immunisation of mice with coumarin-THG and 7-hydroxycoumarin-
THG 111
ix
3.2.4 Preparation of splenomic RNA 113
3.2.5 Selection of murine PCR primers based on homology plots 113
3.2.6 Murine PCR Primers 114
3.2.7 PCR optimisation 117
3.2.8 Preparation of pComb3 for heavy chain insert ligation 119
3.2.9 Measurement of the Electrocompetence of Escherichia coli XLl-Blue 119
3.2.10 Insertion of mouse 5 heavy chain genes into pGEM-T 119
3.2.11 Insertion of mouse 5 variable light chain genes into pGEM-T 120
3.3 DISCUSSION 124
3.3.1 Preparation of protein conjugates for immunisation 124
3.3.2 Characterisation of coumarin-thyroglobulin (coumarin-THG) 125
3.3.3 Mouse serum antibody titres following immunisation 125
3.3.4 PCR primer production 126
3.3.5 PCR optimisation 127
3.3.6 PCR fragment purification 128
3.3.7 Construction of light and heavy chain gene libraries in pGEM-T 128
3.3.8 Heavy and light chain gene insertion into pComb3 128
4.0 THE PRODUCTION OF SINGLE CHAIN Fv (scFv) ANTIBODIES TO
COUMARIN AND 7-HYDROXYCOUMARIN USING THE N1SSIM
LIBRARY 130
4.1 INTRODUCTION 131
4.1.1 Use of the Nissim library in the isolation of single chain scFv (scFv)
antibodies 131
4.1.2 Affinity selection strategies 131
4.1.3 Affinity selection of antibodies from libraries using BIAcore™ 134
4.1.4 Induction of protein expression from phagemid pHENl 135
4.1.5 Screening of antibody clones from phage display libraries 135
x