Antibody Engineering Volume 1

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.

Cloning of the Antigen-binding Site from Hybridoma.- Cloning of Variable Domains from Mouse Hybridoma by PCR.- Coning Hybridoma cDNA by RACE.- Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.- Generation of Antibody Repertoires.- Mouse Immune Libraries for the Generation of ScFv Fragments Directed Against Human Cell Surface Antigens.- Human Antibody Gene Libraries.- Synthetic Antibody Libraries.- Immune Libraries from Nonhuman Primates (NHP).- Generation of Rabbit Immune Libraries.- Selection of Antibody Fragments from Combinatorial Libraries.- Immunotube Selections.- Phage Display and Selection in Microtitre Plates.- Phage Display and Selections on Biotinylated Antigens.- Phage Display and Subtractive Selection on Cells.- Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells.- Improving Phage Display Throughput by Using Hyperphage, Miniaturized Titration and pVIII (g8p) ELISA.- Yeast Display and Selections.- The Generation of Transgenic Mice Expressing Human Antibody Repertoires.- Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay.- Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries.- Engineering and Production of Immunoglobulins.- Aspects of Isotype Selection.- Generation of Heavy and Light Chains (Chimeric Antibodies).- Humanising Antibodies by CDR Grafting.- Humanization by Resurfacing.- Human Antibodies by Guided Selection.- In Silico De-Immunization.- Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis.- Affinity Maturation by Random Mutagenesis and Phage Display.- Engineering of the Fc Region for Improved PK (FcRn Interaction).- Antibody-Dependent Enzyme Prodrug Therapy (ADEPT).- Production of Recombinant Human IgG Antibodies in the Baculovirus Expression System.- Expression of IgA Molecules in Mammalian Cells.- Antibody Characterization.- Expression of Complete Antibodies in Transgenic Plants.- Expression ofFull Length Monoclonal Antibodies (mAb) in Algal Chloroplast.- Expression of IgG Antibodies in Mammalian Cells.- Protein A/G Chromatography.- Epitope Analysis Using Synthetic Peptide Repertoires Prepared by SPOT Synthesis Technology.- Epitope Mapping by Printed Peptide Libraries.- Antibody Epitope Mapping Using Yeast Display.- Size Exclusion Chromatography.- Structural Characterization of Antibodies by Mass Spectrometry.- Antibody Glycans Characterization.- Affinity Measurements by Competition ELISA.- Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis.- Affinity Measurements Using Quartz Crystal Microbalance (QCM).- Affinity Measurements with Radiolabeled Antibodies.- Neutralization Tests.- Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo.- Competitive ELISA.- Quantification of Human IgG and Related Fc Fusion Proteins by a Human IgG/Fc Capture ELISA.- Determination of Fc-Mediated Antibody-Effector Functions by Chromium Release Assay.- Binding Studies with Flow Cytometry.