Monoclonal Antibodies: A Practical Approach

Front Cover
Philip Shepherd, Christopher J. Dean
Oxford University Press, 2000 - Medical - 479 pages
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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Contents

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
Preparation of linker
37
Assembly of VH and VK gene fragments with linker DNA
39
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
72
96
Troubleshooting
107
Cryopreservation of cells
122

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
The design of the humanized antibody
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
72
Choice of number of selection rounds
73
Laboratory based methods for small scale production of monoclonal
125
Isolation and purification of monoclonal antibodies from tissue
149
Antibody production in plants
181
Nonradioactive antibody probes
237
Immunogold probes for light and electron microscopy
247
Characterization of cellular antigens using monoclonal antibodies
265
Immunoassays
297
Immunoaffinity chromatography of macromolecules
319
Immunochemical detection of BrdUrd labelled nuclei for monitoring cell
341
Immunofluorescence microscopy
355
FACS analysis of clinical haematological samples in transplantation
371
Immunocytochemical staining of cells and tissues for diagnostic
391
Detection of chemically modified DNA in lymphocytes of patients undergoing
411
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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