Antibody Engineering: Methods and Protocols

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Springer Science & Business Media, 2004 - 561 Seiten
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A core collection of diverse cutting-edge techniques for the generation, expression, optimization, and characterization of recombinant antibodies. Readily reproducible protocols for lead generation range from the cloning of human immunoglobulin genes to the selection and generation of human recombinant antibodies by humanization approaches, molecular display technologies and transgenic animals. Procedures are also described on restructuring antibody leads into monovalent, multivalent, and bispecific binding fragments for a wide variety of in vivo applications. State-of-the-art technologies are described for the characterization of antigen-binding affinity and specificity with novel applications in radioimmunotargeting, cancer immunotherapy, drug abuse, and proteomics. Use cutting-edge techniques for generating and optimizing recombinant antibodies Generate antibodies by humanization, molecular display technologies and transgenic mice Perform epitope mapping with mass spectrometry, bioinformatics, and array technologies Learn about novel applications in cancer, drug addiction, and proteomics Express recombinant antibodies in bacterial, yeast, insect, mammalian and plant systems.
 

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Inhalt

Internet Resources for the Antibody Engineer
3
The Kabat Database and a Bioinformatics Example
11
IMGT The International ImMunoGeneTics Information System httpimgtcinesfr
27
Antibody Variable Regions Toward a Unified Modeling Method
51
Studying Antibody Conformations by Ultracentrifugation and Hydrodynamic Modeling
93
ANTIBODYLEAD GENERATION
116
PCR Cloning of Human Immunoglobulin Genes
117
Antibody Humanization by CDR Grafting
135
Directed Mutagenesis of Antibody Variable Domains
319
Antibody Affinity Maturation by Chain Shuffling
327
Antibody Affinity Maturation by Random Mutagenesis
345
Developing a Minimally Immunogenic Humanized Antibody by SDR Grafting
361
ANTIBODY CHARACTERIZATION AND NOVEL APPLICATIONS
378
Antibody Purification by Column Chromatography
379
Affinity Measurement Using Surface Plasmon Resonance
389
Kinetic Exclusion Assays to Study HighAffinity Binding Interactions in Homogeneous Solutions
417

Selection of Human Antibodies from Phage Display Libraries
161
Production of Human SingleChain Antibodies by Ribosome Display
177
Production of Human Antibodies from Transgenic Mice
191
Selection of Internalizing Antibodies for Drug Delivery
201
Engineering Multivalent Antibody Fragments for In Vivo Targeting
209
Production of Recombinant Bispecific Antibodies
227
ANTIBODY EXPRESSION AND OPTIMIZATION
244
Expression and Isolation of Recombinant Antibody Fragments in E coli
245
Expression of Recombinant Antibodies in Mammalian Cell Lines
255
Human Antibody Production Using InsectCell Expression Systems
269
Antibody Production in Transgenic Plants
301
Characterization of Antibody Antigen Interactions by Fluorescence Spectroscopy
431
Antibody Epitope Mapping Using Arrays of Synthetic Peptides
443
Mapping Antibody Antigen Interactions by Mass Spectrometry and Bioinformatics
465
Radiometal Labeling of Antibodies and Antibody Fragments for Imaging and Therapy
481
Production and Characterization of AntiCocaine Catalytic Antibodies
495
Recombinant Immunotoxins in the Treatment of Cancer
503
Antibodies in Proteomics
519
Targeting of Antibodies Using Aptamers
547
Index
557
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