Monoclonal Antibodies: A Practical Approach

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Philip Shepherd, Christopher J. Dean
Oxford University Press, 2000 - Medical - 479 pages
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Monoclonal Antibodies: A Practical Approach covers the preparation, testing, derivation, and applications of monoclonal antibodies. New immunological techniques incorporating tried and tested methodologies are described, making the book of interest to established and inexperienced immunologists. Both the standard somatic hybridization technique and recombinant techniques, including the use of phage libraries, for the preparation of rodent and human monoclonal antibodies are described. Protocols for both the small and large scale production are detailed, as well as purification and labelling (with both radioisotopes and non-radioisotopes) methods. The applications of monoclonal antibodies in immunoblotting, enzyme linked immunoassays, immunofluorescence, and FACS analysis are all covered in detail. Finally protocols are given for the use of monoclonal antibodies in rheumatoid arthritis, tissue typing, detecting DNA modified during chemotherapy, and in the clinical analysis of transplantation samples for malignancy. This book will therefore be an invaluable laboratory companion to anyone using monoclonal antibodies in their research.
 

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prepare AG for immunization
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Contents

Screening hybridoma culture supernatants for specific antibody
12
Cloning of hybridomas
18
Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting
25
Preparation of mouse spleen
26
Isolation of total RNA from spleen
27
PolyA mRNA isolation
30
Reverse transcriptase reaction
31
Primary PCR of antibody genes
33
Selection of phage libraries on biotinylated antigen
74
Selections using biotinylated antigen
75
Proximity selections
78
ARM complexes for in vitro display and evolution of antibody
91
Ribosome display methodology
93
Troubleshooting
107
Cryopreservation of cells
122
Laboratory based methods for small scale production of monoclonal
125

Preparation of linker
37
Assembly of VH and VK gene fragments with linker DNA
39
Assembly of single chain Fv antibody fragments
41
Reamplification of assembled scFv DNA
43
Restriction enzyme digestion of assembled scFv
44
Purification of pHEN1 vector by equilibrium centrifugation in CSCI ethidium bromide gradients
46
Restriction digestion of the phage display vector pHEN1
49
Ligation of pHEN1 and insert antibody scFv
51
Preparation of electroporation competent E coli TG1 strain cells
52
Electroporation
53
Analysis of recombinant clones from the library
55
References
57
Appendix
58
Issues to consider
59
References
64
Selection of antibodies from phage libraries of immunoglobulin genes
67
Preparation and storage of phage library stocks
68
Phage libraries
69
Selection of phage libraries on purified immobilized antigen
71
Elution conditions Storage and rescue of the phagemid population after selection
72
Choice of number of selection rounds
73
Summary
145
Antibody production in plants
181
Radiolabelling of monoclonal antibodies
207
Nonradioactive antibody probes
237
Labelling with biotin
243
Introduction
265
Immunoassays
297
Biotin labelling
305
Immunoaffinity chromatography of macromolecules
319
Monitoring of coupling efficiency
329
Analysis of purified antigen
336
Immunofluorescence microscopy
355
FACS analysis of clinical haematological samples in transplantation
371
Analysis of lymphocyte subsets in peripheral blood and bone marrow harvests from
383
Immunocytochemical staining of cells and tissues for diagnostic
391
Biotinylated tyramine
406
Monoclonal antibody therapy in organ transplantation
431
Monoclonal antibody therapy in rheumatoid arthritis
449
A1 List of suppliers
463
Index
469
Copyright

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About the author (2000)

Phil S. Shepherd is at The Guy's, King's College, and St Thomas's Hospitals, London.

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