Immunological Techniques Made Easy

by ; ; ;
Edition: 1st
Format: Paperback
Pub. Date: 1998-08-17
Publisher(s): Wiley
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Summary

Immunological Techniques Made Easy Edited by Olivier Cochet, Biotechnology and Antibody Laboratory, Jean-Luc Teillaud and Catherine Saut?s, INSERM Laboratory of Cellular and Clinical Immunology, Institut Curie, Paris, France. Here, at last, is a clear and concise guide to 100 of the most commonly used immunological techniques that can easily be performed by non-immunologists, and which assumes no prior knowledge of the techniques described. The idea for this book arose from the authors' observations that scientists in many fields of biomedical research needed, at some time or another, to perform an immunological technique applied to their own specific field of research. Existing manuals of immunological techniques are intended primarily for research immunologists and are either too detailed or assume background expertise that the user may not necessarily possess. Each technique is described step-by-step, in an easy-to-follow format, much like a cooking recipe, and is abundantly illustrated to give the user a clear understanding of what is happening at each stage. The book is edited by three experienced immunologists from the Curie Institute in Paris who have brought together an international panel of contributors, all of whom have hands-on expertise of the techniques they describe. Conveniently spiral-bound for easy use at the laboratory bench, the book will be a valuable resource for scientists who want a readily accessible reference to be able to perform immunological techniques successfully and painlessly.

Author Biography

Olivier Cochet is the editor of Immunological Techniques Made Easy, published by Wiley.

Jean-Luc Teillaud is the editor of Immunological Techniques Made Easy, published by Wiley.

Catherine Sautès is the editor of Immunological Techniques Made Easy, published by Wiley.

Table of Contents

List of contributors ix(4)
Foreword xiii(2)
Introduction xv(2)
Abbreviations and symbols xvii
PART ONE CELLULAR IMMUNOLOGY TECHNIQUES 2(112)
Section 1. Cell preparation
1. Peripheral blood mononuclear cell (PBMC) isolation
2(2)
2. Polymorphonuclear cell (PMNC) isolation
4(2)
3. T cell purification
6(4)
4. Monocyte isolation
10(2)
5. Natural killer (NK) cell preparation
12(2)
6. B cell purification from tonsils
14(2)
7. Positive selection of CD34(+) progenitor cells from cord blood
16(2)
8. Cell purification using magnetic beads
18(2)
9. Isolation and enrichment of human epidermal Langerhans cells
20(4)
10. In vitro generation of dendritic cells
24(2)
11. Generation of T cell clones
26(4)
12. Culture of lympokine activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL)
30(2)
13. Generation of human natural killer (NK) cell clones
32(4)
14. CD40 activation of B lymphocytes
36(2)
Section 2. Cell typing
15. Membrane immunofluorescence and cocapping
38(4)
16. Immunofluorescence for intracellular antigens
42(4)
17. Detection of FcGamma receptors (FcGammaR) by rosette assay
46(2)
18. Immunohistology
48(4)
Section 3. Cytotoxicity
19. Complement-dependent cell cytotoxicity
52(4)
20. Cell-mediated cytotoxicity assay
56(4)
Section 4. Cell culture
21. Mononuclear cell activation with mitogens
60(2)
22. Measurement of endotoxin level
62(4)
23. Removal of endotoxins
66(2)
24. Mycoplasma detection
68(4)
25. Mycoplasma removal
72(2)
26. Cell freezing and thawing
74(2)
Section 5. Cell activation
27. Calcium flux
76(4)
28. Protein tyrosine phosphorylation
80(4)
29. Measurement of superoxide release
84(2)
30. Inflammatory mediator release
86(4)
31. Cytokine detection and quantification
90(2)
32. Cytokine mRNA detection
92(4)
Section 6. Cell proliferation
33. Measurement of cell proliferation by [(3)H]thymidine uptake
96(4)
34. MTT assay
100(2)
35. Use of flow cytometry for measurement of T cell proliferation
102(2)
Section 7. Apoptosis evaluation
36. Internucleosomal DNA fragmentation analysis
104(2)
37. Apoptosis evaluation by flow cytometry: the TUNEL technique
106(4)
38. Apoptosis evaluation by Annexin V-FITC
110(4)
PART TWO IMMUNOCHEMICAL TECHNIQUES 114(130)
Section 8. Antigen preparation
39. Coupling peptides with carrier proteins
114(2)
40. Preparation of immunogens and methods for increasing immunogenicity
116(2)
41. Liposome preparation
118(2)
42. Coupling peptides to liposomes
120(2)
43. Cell extract preparation
122(2)
44. Biosynthetic cell labeling
124(2)
Section 9. Generation of polyclonal antibodies
45. Immunization routes
126(2)
46. Bleeding and serum preparation
128(4)
Section 10. Generation of monoclonal antibodies and hybridomas
47. Monoclonal antibody production: general considerations
132(2)
48. Culture of murine mutant fusion partners
134(4)
49. Cell fusion: obtaining B cell hybridomas
138(4)
50. Selection of positive hybridoma clones and cloning by limiting dilution
142(4)
51. Production of human monoclonal antibodies by Epstein-Barr virus (EBV) transformation
146(2)
52. In vitro cell immunization
148(4)
53. Laboratory-scale antibody production
152(4)
54. Selection of switch variants
156(4)
Section 11. Antibody characterization
55. Indirect ELISA by antibody capture
160(4)
56. Quantifying antigens by direct competition assay
164(4)
57. Quantifying antigens by ELISA sandwich assay
168(4)
58. Isotype determination of antibodies by sandwich ELISA
172(4)
59. Detection of specific antibodies by double-immunodiffusion
176(4)
60. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
180(4)
61. Western blotting
184(4)
62. Immunoprecipitation
188(4)
63. Determination of antibody affinity constant (K(aff))
192(4)
64. Measurement of antibody affinity by solid-phase assay
196(4)
65. Kinetic measurements by surface plasmon resonance (SPR)
200(4)
Section 12. Antibody purification
66. Ammonium sulfate precipitation
204(4)
67. Antibody purification by caprylic acid precipitation
208(2)
68. Antibody purification by hydroxylapatite chromatography
210(2)
69. Antibody purification by diethyl amino ethyl (DEAE) ion exchange chromatography
212(2)
70. Coupling antibodies to activated Sepharose 4B beads
214(2)
71. Purification of IgG by protein A or protein G affinity chromatography
216(4)
72. Antigen affinity chromatography
220(2)
73. Antigen purification by antibody affinity chromatography
222(4)
Section 13. Biochemical antibody engineering
74. Antibody labeling with (125)iodine
226(4)
75. Biotinylation
230(2)
76. Fluorescein isothiocyanate (FITC) coupling
232(2)
77. Coupling enzymes to antibodies
234(2)
78. Preparation of F(ab)(2) from IgG using proteolytic enzymes
236(4)
79. Preparation of Fab from F(ab)(2)
240(4)
PART THREE LABORATORY HOSPITAL TECHNIQUES 244(50)
80. Autoantibody detection
244(2)
81. Immune complexes (IC) detection
246(4)
82. Hemagglutination
250(4)
83. Hypergammaglobulinemia and hypogammaglobulinemia detection
254(4)
84. Cryoglobulins (CG) detection and classification
258(4)
85. Monoclonal gammapathies detection by immunoelectrophoresis (IEP)
262(4)
86. Monoclonal gammapathies detection by electroimmunofixation (EIF)
266(4)
87. CD typing by immunofluorescence
270(4)
88. HLA class I serological typing
274(4)
89. HLA class II molecular typing by PCR-SSO
278(4)
90. HLA-DRB DQB1 molecular typing by PCR-SSP RFLP
282(2)
91. HLA-DPB1 molecular typing by PCR-SSP RFLP
284(2)
92. Detection of complement levels by hemolytic assay
286(4)
93. Hemolytic assay of a single component of complement
290(4)
PART FOUR APPENDICES 294(31)
A1. The most common buffers
294(6)
A2. Proteases and protease inhibitors
300(2)
A3. Amino acids and genetic code
302(2)
A4. Protein quantification
304(4)
A5. SDS-PAGE gel staining and drying
308(4)
A6. Substrates for immunoenzymatic reactions
312(4)
A7. Culture media, sera, and additives
316(4)
A8. Sites of interest on the World Wide Web
320(5)
Index 325

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