Antibody Engineering

by Kontermann, Roland; Dubel, Stefan

• ISBN13: 9783642011436
• ISBN10: 3642011438
• Edition: 2nd
• Format: Hardcover
• Copyright: 2010-05-07
• Publisher: Springer Verlag

Summary

Antibodies are indispensable tools for research, diagnosis, and therapy. Recombinant approaches allow the modification and improvement of nearly all antibody properties, such as affinity, valency, specificity, stability, serum half-life, effector functions, and immunogenicity.Antibody Engineering provides a comprehensive toolbox covering the well-established basics but also many exciting new techniques. The protocols reflect the latest "hands on" knowledge of key laboratories in this still fast-moving field. Newcomers will benefit from the proven step-by-step protocols, which include helpful practical advice; experienced antibody engineers will appreciate the new ideas and approaches. The book is an invaluable resource for all those engaged in antibody research and development.

Table of Contents

Cloning of the Antigen-binding Site from Hybridoma
Cloning of Variable Domains from Mouse Hybridoma by PCR	p. 3
Coning Hybridoma cDNA by RACE	p. 15
Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly	p. 21
Generation of Antibody Repertoires
Mouse Immune Libraries for the Generation of ScFv Fragments Directed Against Human Cell Surface Antigens	p. 47
Human Antibody Gene Libraries	p. 65
Synthetic Antibody Libraries	p. 85
Immune Libraries from Nonhuman Primates (NHP)	p. 99
Generation of Rabbit Immune Libraries	p. 115
Selection of Antibody Fragments from Combinatorial Libraries
Immunotube Selections	p. 127
Phage Display and Selection in Microtitre Plates	p. 139
Phage Display and Selections on Biotinylated Antigens	p. 151
Phage Display and Subtractive Selection on Cells	p. 165
Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells	p. 183
Improving Phage Display Throughput by Using Hyperphage, Miniaturized Titration and pVIII (g8p) ELISA	p. 197
Yeast Display and Selections	p. 207
The Generation of Transgenic Mice Expressing Human Antibody Repertoires	p. 235
Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay	p. 255
Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries	p. 267
Engineering and Production of Immunoglobulins
Aspects of Isotype Selection	p. 291
Generation of Heavy and Light Chains (Chimeric Antibodies)	p. 307
Humanising Antibodies by CDR Grafting	p. 319
Humanization by Resurfacing	p. 341
Human Antibodies by Guided Selection	p. 355
In Silico De-Immunization	p. 369
Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis	p. 377
Affinity Maturation by Random Mutagenesis and Phage Display	p. 397
Engineering of the Fc Region for Improved PK (FcRn Interaction)	p. 411
Antibody-Dependent Enzyme Prodrug Therapy (ADEPT)	p. 431
Production of Recombinant Human IgG Antibodies in the Baculovirus Expression System	p. 453
Expression of IgA Molecules in Mammalian Cells	p. 471
Antibody Characterization
Expression of Complete Antibodies in Transgenic Plants	p. 489
Expression of Full Length Monoclonal Antibodies (mAb) in Algal Chloroplast	p. 503
Expression of IgG Antibodies in Mammalian Cells	p. 517
Protein A/G Chromatography	p. 531
Epitope Analysis Using Synthetic Peptide Repertoires Prepared by SPOT Synthesis Technology	p. 537
Epitope Mapping by Printed Peptide Libraries	p. 573
Antibody Epitope Mapping Using Yeast Display	p. 591
Size Exclusion Chromatography	p. 607
Structural Characterization of Antibodies by Mass Spectrometry	p. 613
Antibody Glycans Characterization	p. 635
Affinity Measurements by Competition ELISA	p. 657
Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis	p. 667
Affinity Measurements Using Quartz Crystal Microbalance (QCM)	p. 683
Affinity Measurements with Radiolabeled Antibodies	p. 695
Neutralization Tests	p. 705
Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo	p. 723
Competitive ELISA	p. 739
Quantification of Human IgG and Related Fc Fusion Proteins by a Human IgG/Fc Capture ELISA	p. 743
Determination of Fc-Mediated Antibody-Effector Functions by Chromium Release Assay	p. 749
Binding Studies with Flow Cytometry	p. 765
Index	p. 781
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