Generation and Optimisation of Novel Recombinant Antibodies for Improved Cardiac Diagnostics

350 Pages·2011·13.63 MB·English

Generation and Optimisation
of Novel Recombinant Antibodies
for Improved Cardiac Diagnostics
A thesis submitted for the degree of Ph.D.
by
B. Vijayalakshmi Ayyar M.Sc.,
Based on the research carried out at the
School of Biotechnology
and the
Biomedical Diagnostics Institute,
Dublin City University,
Dublin 9, Ireland.
Under the supervision of Professor Richard O’Kennedy
March, 2011.
Declaration:
I hereby certify that this material, which I now submit for assessment on the programme
of study leading to the award of Doctor of Philosophy is entirely my own work, that I
have exercised reasonable care to ensure that the work is original, and does not to the
best of my knowledge breach any law of copyright, 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 work.
Signed: ____________________ (Candidate) ID No.: 56124074 Date: __________
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ACKNOWLEDGEMENTS
I would like to take this opportunity to thank all people who have helped me with
important contributions, through advice and encouragement during my doctoral study.
I must first and foremost give my gratitude to my supervisor, Professor Richard
O’Kennedy, for his incisive guidance, boundless patience and constant encouragement.
My deepest thanks are also extended to internal examiner Professor Colette McDonagh
forhervaluable opinions on my thesis.
I would like to thank Dr. Stephen Hearty for supervising my project and helping me
with the timely completion of my project.
I must thank the Biomedical Diagnostics Institute (BDI) for providing financial
assistanceduring this project.
I would like to acknowledge the support and inputs I received from all my lab buddies
at theApplied Biochemistry Group (ABG).
I wish to thank all my friends here in Dublin who helped me get through these four
years of my doctoral research.
A special acknowledgement goes to Sushrut, a good friend, who was always there
beside me whenever I needed him.
I am indebted to my family (my late father, my family, my younger brother, and my
grandmother) for their unfaltering love and support. I am especially indebted to my late
father, who worked industriously to support my education, but who never lived long
enough to see me through to the end of this study. He will always be the source of my
inspiration and a part of me.
Finally, my greatest regards go to the Almighty for making me worthy of what I am
today.
TABLE OF CONTENTS
Declaration ii
Dedication iii
Acknowledgements iv
Table ofContents v
Abstract xi
List ofFigures xii
List ofTables xv
Abbreviations andAcronyms xvii
Units xxi
Publications andPresentations xxii
1. Introduction 1
1.1 Cardiovascular diseases 2
1.1.1 Introduction tocardiovascular diseases 2
1.1.2 Pathophysiology 4
1.1.3 Acute myocardial infarction 9
1.2 Biomarkers 10
1.2.1 Emergence of biomarkers as diagnostic tools in cardiovascular disease 11
1.2.2 Characteristics of cardiacbiomarkers 13
1.2.3 Markers of ischemia and necrosis 14
1.2.4 Heart-fatty acid binding protein 17
1.2.5 Cardiac troponinI 19
1.3 Antibodies 22
1.3.1 Antigens and antibodies 22
1.3.2 Different classes of antibodies 23
1.3.3 Antibody structure 24
1.3.4 Characteristics of antibodies 26
1.3.5 Types of antibodies and their production 28
1.3.6 Recombinant antibody libraries, construction and selection 33
1.3.7 Phage display 36
1.3.7.1Biology of phage display 38
1.3.8 Recombinant antibody formats 43
1.4 Surface plasmon resonance in antibody characterisation 45
1.5 Aims 50
2. Materials andMethods 51
2.1 Materials 52
2.1.1 Equipment 52
2.1.2 Culture compositions 53
2.1.3 Bacterial strains 54
2.1.4 Buffer compositions 54
2.1.5 Commercial kits 57
2.1.6 Commercial antigens and antibodies 58
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2.2 Methods 59
2.2.1 Generation of chicken anti-hFABP scFv 59
2.2.1.1 Immunisation of chicken with hFABP 59
2.2.1.2 RNA extraction and cdna preparation 60
2.2.1.3 Reverse transcription of total RNA to cDNA 62
2.2.1.4 PCR primers for amplification of chicken scFv (pComb series) 63
2.2.1.5 Amplification of antibody variable domain genes using pComb series primer 64
2.2.1.6 Purification of V and V variablegene-fragments usingQiagen gel
H L
extraction kit 65
2.2.1.7 Splice-by-overlap extension (SOE) PCR 65
2.2.1.8 SfiIrestriction digestionof purifiedSOE-PCRfragment and ligation into
pComb3XSSvector 66
2.2.1.9 Transformation of cloned gene intoE. coliby electroporation 68
2.2.1.10 Rescue and subsequent precipitation of scFv-displaying phage 69
2.2.1.11 Selection and enrichment of hFABP clonesviabiopanning 70
2.2.1.12 Polyclonal phage ELISA and colony pick PCR 71
2.2.1.13 Infecting pan 4 output phage to Top10F′ cells for soluble expression 72
2.2.1.14 Titration of scFvs and inhibition ELISA 73
2.2.1.15 Preconcentration study of mouse anti-HA monoclonalantibody onto a
CM5 sensor chip 74
2.2.1.16 Preconditioning and immobilisation of mouse anti-HA monoclonal
antibody on a CM5sensor chip surface 75
2.2.1.17 Ranking of clones by ‘off-rate’ analysis using Biacore 3000 76
2.2.1.18 DNA fingerprinting of the selected clones to check thediversity 77
2.2.1.19 Kinetic analysis of anti-hFABP clones to analyse their binding
affinity to hFABP 79
2.2.1.20 Large-scale production of anti-hFABP scFvs and purificationusing IMAC 79
2.2.1.21 Protein purification analysis by SDS-PAGE 81
2.2.1.22 Cross-reactivity analysis of anti-hFABP scFv with closely related
FABP isoforms 81
2.2.1.23 Inhibition assays using anti-hFABPscFv 83
2.2.1.24 Lateral flow assay using purified chickenanti-hFABP scFv 84
2.2.1.25 Rabbit anti-hFABP polyclonal antibody 85
2.2.2 Expression of human hFABP 86
2.2.2.1 PCR primers for amplification of hFABP 86
2.2.2.2 Amplificationof hFABPgenefrom human heartcDNA 86
2.2.2.3 Digestion and ligationofhFABPgene into pET-26b(+) vector 87
2.2.2.4 Transformation of cloned gene intoE. colicells 88
2.2.2.5 Growing transformants and protein expression 88
2.2.2.6 SDS-PAGE and Western blot analysis of the expressed proteins 89
2.2.3 Epitope mapping of chicken anti-hFABP antibody 90
2.2.3.1 Epitope mapping using commercial peptide library 90
2.2.3.1.1 Depletion of library against capture antibody-coatedmagneticbeads 90
2.2.3.1.2 Selection and enrichment of anti-hFABP scFv binding peptides 91
2.2.3.1.3 Rescue and subsequent precipitation of peptide-displaying phage 91
2.2.3.1.4 DNA isolation from plaques and sequencing 93
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2.2.3.2 Epitope mappingusing gene-fragment library 94
2.2.3.2.1 Modification of vector 94
2.2.3.2.2 Preparation of chemically competent cells 96
2.2.3.2.3 Transformation of cloned HCS into theE. coliand screeningof
transformed clones 97
2.2.3.2.4 OptimisingDNaseI digestion of hFABP gene 99
2.2.3.2.5 Modifying the gene-fragments for cloning into the vector 100
2.2.3.2.6 Cloning the gene-fragments into the vector and transforming it
intoE. colicells 102
2.2.3.2.7 Changing anti-hFABP (3.G9) scFv into scAb 103
2.2.3.2.8 Biopanning of gene-fragment library 104
2.2.3.2.9 Polyclonal phage ELISA of gene library panned outputs 106
2.2.3.3 Gene walking 106
2.2.3.3.1 Cloning and expressionof hFABP gene-fragments 108
2.2.4 Rabbit anti-cTnIscFv library 110
2.2.4.1 Immunisation of rabbit withcTnIand determining the serumtitre 110
2.2.4.2 Characterisation of rabbit serum against different cTnIepitopes 110
2.2.4.3 Anti-cTnIpolyclonal antibody purification from rabbit serum 111
2.2.4.4 CheckerboardELISAof purified anti-cTnIpolyclonal antibody 112
2.2.4.5 RNAextraction and cDNApreparation 112
2.2.4.6 PCRprimers for amplification of rabbit scFv (pComb series) 112
2.2.4.7 Amplification of antibody variable genes using pComb3Xseries primers 116
2.2.4.8 Splice-by-overlap extension (SOE) PCR 117
2.2.4.9 Digestionof vector and purifiedSOE-PCRproduct and cloning 118
2.2.4.10 Transformation of cloned gene intoE. colibyelectroporation 119
2.2.4.11 Rescueand subsequent precipitation of scFv-displaying phage 119
2.2.4.12 Biopanning the library against cTnIfor enrichment ofspecific clones 120
2.2.4.13 Polyclonal phageELISAand soluble expression of pan 5 outputs 121
2.2.4.14 Screening cTnIclones by indirectELISA 121
2.2.4.15 Cross-reactivity analysis of positive clones withskeletal troponinI(sTnI) 122
2.2.4.16 Competitiveanalysis of cTnIpositive clones 122
2.2.4.17 Rankingof clones by ‘off-rate’ analysis usingBiacore 3000 123
2.2.4.18 Restrictionmapping of the clones 123
2.2.4.19 Cloningand expression ofcTnIgene into pET-32b(+) vector 123
2.2.4.20 SDS-PAGEandWesternblotanalysis ofthe lysates 125
2.2.5 Formattingof rabbit anti-cTnIantibodies for optimal expressionand conjugation 125
2.2.5.1 Modificationof pMoPac vector 125
2.2.5.1.1 Primersfor amplification of rabbit and chickenconstant regions 126
2.2.5.1.2 Amplificationof rabbit and chicken constant regions 126
2.2.5.1.3 Digestionand cloning of the constant regions intopMoPac vector
and itstransformation inE. colicells 127
2.2.5.2 Changingvariable domain orientation 129
2.2.5.2.1 Primersfor reversing the orientation 130
2.2.5.2.2 Amplificationof heavy and light variable domains 130
2.2.5.2.3 SOE-PCRfor assembling the variable domains 131
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2.2.5.3 ChangingtheSfiIsites in the primers for intercloning intopComb and
pMoPacvectors 132
2.2.5.3.1 Primersfor changing theSfiIsites for cloninginto differentvectors 132
2.2.5.3.2 AmplificationofSOEproducts for cloning intodifferent vectors 132
2.2.5.4 Cloningof scFv gene with different orientation andSfiIsitesinto pComb
and pMoPacvectors 133
2.2.5.5 Expressionanalysis of anti-cTnIantibody in different formats 135
2.2.5.6 Purificationanalysis of anti-cTnIantibody in differentformats 136
2.2.5.7 Site-directed mutagenesis of the MG4 scFv 136
2.2.5.7.1 Primersfor site-directed mutagenesis ofMG4 scFv 137
2.2.5.7.2 Site-directed mutagenesis ofCys 80 toSer 80 andAla 80 byPCR 137
2.2.5.7.3 Transformationand screening of clones 139
2.2.5.7.4 Expressionof mutated scFvs withMG4 139
3. Generation of aChickenAnti-hFABP 140
3.1 Introduction 141
3.1.1 Chickenas a host 141
3.1.2 Heart-fatty acid binding proteinas an analyte 143
3.2 Construction of chicken anti-hFABPscFv library and enrichment of specific clonesviabiopanning 144
3.3 Screeningof hFABP-specific clones 145
3.4 Rankingof clones byBiacore3000 148
3.4.1 Preparationof chip for ranking analysis 148
3.4.2 Immobilisationof capture antibody 151
3.4.3 ‘Off-rate’ analysis of clones 154
3.4.4 Kineticanalysis of scFvs 159
3.5 Sequence analysis of scFvs 164
3.6 Cross-reactivity analysis of thescFvs 165
3.7 Inhibitionassays 167
3.8 Lateral flow immunoassay 169
3.9 Rabbit polyclonal serum titre 170
3.10 Discussion 172
4. Expression ofHumanHeart-Fatty Acid Binding Protein 178
4.1 Introduction 179
4.1.1 E.colias an expression system 179
4.2 Amplification of hFABPfrom human heart cDNA 180
4.3 Cloning of hFABPgene in pET-26b(+) vector 181
4.4 Transformation of the cloned gene inE. colicells and expression of protein 182
4.5 Analysis of the hFABPexpressing clones by sequencing 184
4.6 Large-scale protein expression andIMACpurification of the expressedprotein 185
4.7 Discussion 187
5. EpitopeMapping ofAnti-hFABP scFv 190
5.1 Introduction 191
5.2 Epitope mapping using Ph.D.™-12 Peptide Library 195
5.2.1 Selection of anti-hFABP scFv specific peptides 195
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5.2.2 Screening of anti-hFABP scFv specific peptides 196
5.2.3 Sequencing of peptides selected after panning 197
5.3 Epitope mapping using gene-fragment library 199
5.3.1 Construction of gene-fragment library 199
5.3.2 Modification of vector 199
5.3.3 Generation of gene-fragments and cloning into modified pComb3HSS vector 202
5.3.4 Selection and screening of the gene-fragment library 205
5.4 Epitope mapping by recombinant expression of gene-fragments 207
5.4.1 Expression of gene fragments by excluding regions at C-terminal of the hFABP 207
5.4.2 Expression of gene fragments in by excluding regions at N-terminal of the hFABP gene 212
5.5 Discussions 215
6. Generation ofRabbitAnti-cTnI scFv 220
6.1 Introduction 221
6.1.1 Rabbitas a host 221
6.1.2 CardiactroponinIas an analyte 222
6.2 Rabbit immunisation andcTnIserum titres 224
6.3 Purification of rabbit polyclonal antibodies and their analysis bycheckerboardELISA 228
6.4 Optimisation of variable gene amplifications from rabbit cDNA 230
6.5 Rabbit SOE-PCRfor assembling variable heavy and light chain 232
6.6 Libraryconstruction and biopanning 233
6.7 Analysis of biopanning outputs by polyclonal phageELISA 235
6.8 Soluble expression and indirectELISAfor screeningcTnIclones 236
6.9 Cross-reactivity analysis of positive clones withsTnI 237
6.10 Solution phase inhibitionELISAfor selection of scFvs capable of use in a competitive
immunoassay 238
6.11 Ranking ofanti-cTnIscFvs 239
6.12 Fingerprinting analysis of anti-cTnIscFvs 242
6.13 Expression of recombinant cTnI 243
5.13.1 Amplificationof thecTnIgene for expression 243
5.13.2 Genesynthesis, cloning and transformation intoE. colicells 245
5.13.3 AnalysisofcTnIexpression by the transformants 245
6.14 Discussion 247
7. FormattingRabbitAnti-cTnIAntibodies forOptimalExpression 250
7.1 Introduction 251
7.2 Sequencing of rabbit anti-cTnIscFvs 253
7.3 Comparative expression analysis ofanti-cTnIscFvswith different aminoacid at position 80 255
7.4 ReformattingMG4 scFv 257
7.4.1 Modificationof pMoPac vector 257
7.4.2 Changingvariable domain orientation 260
7.4.3 Changingthe restriction sites on theSOE-PCRproduct 262
7.4.4 Transformationand screening of antibodies with different formats 264
7.5 Expression analysis of different antibody formats 266
7.6 Comparative purification analysis of rabbit anti-cTnIantibodies byIMAC 269
7.7 Site-directed mutagenesis ofCys80 onMG4 scFv withV -V domain 272
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7.8 Screening of mutated clones 274
7.9 Comparative expression analysis ofMG4 scFvwith different amino acids at position 80 in
the variablelight chain region 276
7.10 Discussion 277
8. Conclusions 281
9. Bibliography 289
Appendix 1 326
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