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Epitope mapping : a practical approach / edited by Olwyn M. R. Westwood and Frank C. Hay. — Oxford ;New York : Oxford University Press, 2001.—(63.173/E64) |
Contents
Contents
List of protocols page xiii
Abbreviations xvii
1 An introduction to epitope mapping 1
Olwyn M. R. West:wood and Frank C. Hay
1 Environmental conditions can influence protein structure 1
Locating the epitope of the molecule 2
2 Lessons from an historical perspective 4
Enzymatic cleavage for epitope mapping 4
Protein sequence analysis 5
Chemical characteristics of sequences 6
3 Synthetic peptide technologies for epitope mapping 6
4 Chemical modification of antigens 7
5 Site-directed mutagenesis as a tool for epitope mapping 8
6 Hybridoma technology and epitope analysis 9
Protein footprinting in epitope analysis 10
7 Generating monoclonal antibody 10
8 Phage display libraries 10
9 Carbohydrates and their significance when epitope mapping 11
10 Approaches to epitope mapping 13
Polyclonal or monoclonal antibody? 13
Whole antigen available? 13
Amino acid sequence known? 13
Nucleotide sequence available? 13
References 13
2 Multiple Pin Peptide Scanning ("Pepscan") 17
Nazira Sumar
1 Introduction 17
Brief outline of Pepscan 18
2 Solid phase peptide synthesis 18
Attachment 19
Deprotection and coupling 21
Elongation of peptide chain 21
Cleavage 21
3 Multiple peptide synthesis on pins 22
Pins 22
Choice of peptide length 22
4 Testing of antibody epitopes 27
Pin-bound non-cleavable peptides 27
Biotinylated peptides 30
5 Results and data analysis 32
6 Further analysis 33
Window net analysis 33
Replacement net analysis 33
7 Applications of Pepscan 36
8 Other systems for epitope analysis 38
Simple precision original test system (SPOTs) 38
Tea-bag synthesis 38
Peptide epitope libraries 40
Acknowledgements 43
References 43
3 Methodological tips for human T cell epitope mapping when using pin technology peptide arrays 47
David A. Mutch and Olwyn M. R. Westwood
1 Introduction 47
2 Historic foundations to large scale T cell epitope mapping 48
3 Fundamental considerations 49
Lessons from B cell epitope mapping 49
The nature of the peptide antigen 52
Protein sequence databases and sequence analysis 52
4 Peptide synthesis considerations 52
Types of peptides 53
Features of T cell epitopes 53
5 Use of pin peptides in human PBMC proliferation and cell culture assays 59
Buffer systems 61
Cleavage of peptide and peptide concentration 61
Using human cells 62
Cell culture conditions 57
Pulse radiolabelling 75
6 Data analysis using the ALLOC algorithm 76
7 Example of assay conditions for large scale mapping of T cell epitopes Individual steps in the T cell epitope mapping protocol 82
Example of results obtained 84
References 87
Published methods to detect cellular antigenic stimulation 88
4 Combined B cell and T cell epitopes 91
Sowsan F. Atabani
1 Introduction 91
2 Molecular mapping of antigenic and immunogenic epitopes 92
Identification of antigenic epitopes in vitro 92
Identification ofT cell epitopes in vitro 94
3 In vivo analysis of immunogenicity and antigenicity 95
Investigation of the core sequence of B cell and T cell epitopes 97
Inhibition ELISA for the measurement of antibody affinity as a method to determine the B cell epitope 98
Determination of precise helper T cell epitopes by proliferative and cytokine responses 99
4 General summary 101
References 102
5 CTL epitopes 103
Tim EllioVi and John S. Haururn
1 Introduction 103
TcR recognizes a complex between MHC and antigenic peptide 103
Antigen processing 105
Immunodominance 107
2 Indirect methods of CTL epitope identification using synthetic peptides 108
Peptide binding to class I MHC molecules 112
Correspondence between motifs and binding efficacy 118
Correspondence between peptide binding and immunogenicity/immunodominance 120
3 Direct identification of CTL epitopes 123
Direct identification ofCTL epitopes by immuno-isolation and sequencing 123
Direct identification of CTL epitopes by expression cloning 128
References 131
Appendix 1 Class I MHC peptide motifs 136
Appendix 2 Class I MHC alleles expressed by TAP-deficient cell lines 140
Appendix 3 Monoclonal antibodies specific for class I MHC molecules 141
Appendix 4 Index peptides suitable for modification 142
6 The design， synthesis， and characterization of molecular mimetics 143
Ian T. W. Matthews
1 Introduction 143
2 Design， synthesis， and characterization of peptoid oligomers 144
Design of peptoid oligomers 144
The synthesis of peptoid oligomers 147
Characterization of peptoids 150
3 Further oligomeric peptide mimetics 151
β-Peptides，/3-peptoids， retro-peptoids， and amide surrogates 151
4 Non-oligomeric peptide mimetics 155
Natural templates 155
Non-natural templates 156
References 157
7 Generating monoclonal antibody probes and techniques for characterizing and localizing reactivity to antigenic determinants 159
Paul N. Nelson
1 Introduction 159
2 Immunization strategies 160
3 Hybridization and culture of hybridomas 163
4 Monoclonal purification 169
5 Monoclonal conjugation 170
6 Techniques for screening and characterizing mAb reactivity to antigenic determinants 171
Enzyme-linked immunosorbent assay (ELISA) 172
Haemagglutination 176
Slot-blotting 178
Immunocytochemistry 180
7 Physicochemical and chemical modification， pepsin digestion 185
8 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 187
9 Protein determination 194
UV absorption 194
Fluorescence 196
Acknowledgements 197
References 197
8 Epitope mapping of carbohydrate binding proteins using synthetic carbohydrates 199
U. J. Nilsson and G. Magnusson
1 Introduction 199
2 Synthetic carbohydrate analogues 2OO
Oligosaccharide fragments 200
Deoxy analogues 200
Deoxyfluoro analogues 202
O- and C-methyl analogues 202
Amino and carboxy analogues 203
Conformationally restricted or altered analogues 203
Combinatorial synthesis of carbohydrate libraries 203
3 Measuring carbohydrate-protein interactions 204
Haemagglutination inhibition 205
Competitive ELISA 207
Techniques for direct measurement of carbohydrate-protein interaction 215
Enzyme assays 216
Qualitative in situ assays 216
4 Data interpretation 217
Conformational analysis 217
Fragments ofoligosaccharides 218
Deoxy and deoxyfluoro analogues 219
Amino and carboxy analogues 220
Conformationally altered analogues 221
References 222
9 Phage display libraries 225
Samantha Williams， Paul van der Log*， and Volker Germaschewski
1 Introduction 225
General 225
Overview of phage display libraries 227
Applications of phage display libraries 229
2 Case study: identification of dominant epitopes 231
Project strategy 231
Procedure for the identification of peptide epitopes by phage display 231
Deriving peptide binding antibody fragments 243
3 Conclusions 253
Acknowledgements 253
References 253
10 Site-directed mutagenesis in epitope mapping 255
Samuel Perdue
1 Introduction 255
2 General approaches to mutagenesis in epitope mapping: techniques in site-directed mutagenesis 258
Traditional approach 258
PCR approach 263
3 Conclusions 268
References 268
AI List of suppliers 271
Index 277
