Immunochemistry 1 A Practical Approach,9780199636068

Immunochemistry 1 A Practical Approach

by ;
ISBN13: 9780199636068
ISBN10: 0199636060
Format: Hardcover
Pub. Date: 1997-10-02
Publisher(s): Oxford University Press
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Summary

Immunochemistry is of immense importance to virtually all areas of modern biology and medicine, and the space afforded by two volumes has allowed coverage of a wide tange of topics. The familiar and clear Practical Approach format will make a large body of valuable information easilyaccessible to a diverse readership.

Table of Contents

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

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