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Tables of Contents for Immunochemistry 1
Chapter/Section Title
Page #
Page Count
Contributors
xxi
4
Abbreviations
xxv
1. Chemically engineered antibodies
1
26
G. T. Stevenson
K. S. Kan
1. Introduction
1
1
2. Some essential features of IgG molecules
2
1
Interchain SS bonds
2
1
Proteolytic dissections
3
1
3. Some further reactions of SH groups and SS bonds
3
5
Properties of pyridyl disulfides
3
3
SS-interchange on reduced Ig modules
6
1
Alkylation of SH groups for blocking or cross-linking
7
1
4. Buffers and equipment
8
4
Buffers
8
2
Equipment
10
2
5. The human Fcy Module
12
6
Precautions when handling human material
12
1
Basic IgG
12
2
Preparation of Fcy1
14
2
Preparation of Fc-SS-pyridine and Fc-maleimide
16
2
6. Murine Fab'y modules
18
3
IgG antibodies from ascites or culture fluid
18
1
Preparation of F(ab'y)(2)
18
2
Fab'-SS-pyridine and Fab'(-SH)(1)
20
1
7. Some derivatives of Fcy and Fab'y modules
21
3
Chimeric FabFc(2)
21
1
Thioether-linked F(ab')(2) with potential hinge SH groups
22
1
Bispecific chimeric derivatives from thioether-linked F(ab')(2)
23
1
References
24
3
2. Genetically engineered antibodies
27
28
Raymond J. Owens
1. Introduction
27
1
2. PCR cloning of immunoglobulin variable domains
28
7
Synthesis of first strand cDNA
28
2
Primer design
30
3
Problems with the method
33
2
3. The construction of engineered human variable domains
35
6
Designing the sequence
35
3
Assembling the sequence
38
3
4. Construction of Fvs and single-chain Fvs
41
3
Singie-chain Fvs
41
1
Disulfide-linked Fvs
42
2
5. Expression of recombinant antibodies
44
8
Mammalian cells
44
4
E. coli
48
4
Acknowledgements
52
1
References
52
3
3. Production of human monoclonal antibodies from B lymphocytes
55
28
M. D. Melamed
J. Sutherland
1. Introduction
55
1
2. Equipment and media
56
2
Basic requirements
56
1
Media
57
1
Supplements
57
1
3. Assays for detection of human monoclonal antibodies
58
5
ELISA
59
2
Haemagglutination assays
61
2
4. Donor selection
63
1
5. Isolation and proliferation of human B lymphocytes
64
5
Introduction
64
1
Isolation of PBMC from human blood
65
1
Production and use of Epstein-Barr virus
65
1
T cell depletion
66
3
6. Cell fusion
69
4
Introduction
69
1
Fusion of LCLs
70
1
Electrofusion
71
2
7. Strategies for production of human Mabs
73
2
EBV only
73
1
Fusion of selected LCLs
73
1
Fusion of bulk LCLs
74
1
Use of anti-CD40
75
1
8. Cloning
75
4
Limiting dilution
76
2
Other techniques
78
1
Points to note
78
1
9. Scale up of production
79
2
Acknowledgements
81
1
References
81
2
4. Generation and selection of human monoclonal antibodies against melanoma-associated antigens: a model for production of anti-tumour antibodies
83
6
M. D. Thomas
M. D. Melamed
J. Newton
1. Introduction
83
1
2. Production of hybridomas from lymph node lymphocytes
83
1
3. Identification of tumour-specific Mabs
84
4
References
88
1
5. Antibodies packaged in eggs
89
20
Jens Christian Jensenius
Claus Koch
1. Chicken antibodies
89
1
2. Antibodies in eggs
90
1
3. Chicken husbandry
91
1
4. Immunization
91
2
5. The antibody response
93
2
6. Purification of IgG from yolk
95
6
7. Use of chicken antibodies
101
5
References
106
3
6. Standardization of immunochemical procedures and reagents
109
12
Robin Thorpe
Meenu Wadhwa
Tony Mire-Sluis
1. Introduction
109
1
2. Quality of immunochemical reagents
109
1
3. Standardization of immunochemical procedures
110
5
Use of standards
110
1
Nature and availability of standard preparations
111
1
Unitage of standard preparations
112
3
4. Problems with the standardization of immunochemical procedures
115
4
Matrix effects
115
1
Use of multiple standards
115
4
Problems due to the epitope specificity of antibodies
119
1
Acknowledgements
119
1
References
119
2
7. Synthetic peptides in epitope mapping
121
26
Stuart J. Rodda
Gordon Tribbick
N. Joe Maeji
1. Introduction
121
1
2. Multiple peptide synthesis on pins
121
1
Cleaved or non-cleavable?
122
1
Choice of ending
122
1
3. Peptide synthesis
122
5
Storage and handling of peptides
126
1
4. Antibody (B cell) epitope mapping with synthetic peptides
127
7
Direct binding of antibodies on pins
127
3
Direct binding on biotinylated peptides
130
2
Confirmation of relevance of binding
132
2
Analoguing for detailed epitope analysis
134
1
5. T cell epitope mapping with synthetic peptides
134
11
Design of peptide sets
135
3
Epitope mapping with T cell clones
138
3
Mapping of T helper epitopes with peripheral blood mononuclear cells
141
4
Acknowledgements
145
1
References
145
2
8. Enzyme-linked immunoassays
147
30
D. M. Kemeny
1. Introduction
147
1
2. Assay format
147
5
Non-competitive ELISA
147
2
Competitive ELISA
149
3
3. Enzymes and substrates
152
2
Alkaline phosphatase (EC 3.1.3.1)
152
1
Horse-radish peroxidase (EC 1.11.1.7)
153
1
XXX-Galactosidase (EC 3.2.1.23)
154
1
4. Purification of conjugate
154
1
5. Assay optimization
155
1
6. Coating of the solid phase
155
2
7. Sample
157
1
8. Labelled detector
158
1
9. Equipment
159
1
10. Buffers and substrates
160
2
Buffers
160
1
Enzyme substrates
161
1
11. Enzyme labelling of proteins
162
3
12. Assays
165
5
13. Quantification
170
3
Quality controls
172
1
Coefficient of variation
173
1
References
173
4
9. Enzyme amplification systems in ELISA
177
16
Colin H. Self
David L. Bates
David B. Cook
1. Introduction
177
2
2. Enzyme-amplified assay with colorimetric determination
179
5
3. Application of fluorescence to enzyme amplification
184
5
Water sources and buffers
186
1
Preparation of enzymes
187
1
Alkaline phosphatase substrate
187
1
Balance of cycling enzyme concentrations
187
2
4. Concluding remarks
189
1
References
190
3
10. Photoluminescence immunoassays
193
22
I. A. Hemmila
1. Introduction
193
2
2. Lanthanides as probes
195
1
3. DELFIA as a label system for bioanalytical assays
195
16
Microtitration plates as solid phase matrix
197
2
Labelling of immunoreagents with lanthanide ions
199
3
Dissociative fluorescence enhancement of lanthanides and their chelates
202
1
Time-resolved fluorometry of lanthanides
202
2
DELFIA multilabel measurement
204
1
DELFIA FIA and IFMA: assay optimization
205
4
Receptor binding assay with DELFIA technology
209
1
DELFIA cytotoxic release assays
209
2
4. Time-resolved fluorometric assays with stable fluorescent chelates
211
1
5. Conclusion
212
1
References
213
2
11. Enzyme immunoassays for detection of cell surface molecules, single cell products, and proliferation
215
24
N. W. Pearce
J. D. Sedgwick
1. Introduction
215
1
2. Cell-ELISA
216
6
Background to the technique
216
1
Protocol for cell-ELISA
216
5
Advantages and disadvantages
221
1
Applications of the technique
221
1
3. ELISPOT
222
8
Background to the technique
222
1
Protocol for ELISPOT
223
3
Protocol for reverse ELISPOT
226
3
Advantages and disadvantages
229
1
Applications of the technique
230
1
4. Cell proliferation
230
6
Background to the technique
230
1
Protocol for cell proliferation using BrdU
231
1
Advantages and disadvantages
231
5
Applications of the technique
236
1
References
236
3
12. Immunotoxins
239
36
Elaine J. Derbyshire
Claudia Gottstein
Philip E. Thorpe
1. Introduction
239
2
2. Preparation of chemically coupled immunotoxins
241
12
General considerations
241
1
Conjugation methods
242
11
3. Purification of chemically coupled immunotoxins
253
2
Gel permeation chromatography
253
1
Affinity chromatography
254
1
4. Calculation of the composition of chemically prepared ITs
255
1
5. Preparation of ITs by genetic engineering
255
14
Extraction of RNA from B cell hybridoma cell line
256
2
Preparation of DNA coding for variable regions
258
2
Cloning of DNA and assembly of DNA encoding scFv and scFv-immunotoxin
260
6
Expression and purification of fusion proteins
266
3
6. Biochemical characterization of ITs
269
1
Acknowledgements
270
1
References
271
4
A1 List of suppliers
275
8
Index
283