Antibody Engineering Protocols

ISBN-13: 9780896032750 / Angielski / Miękka / 1995 / 450 str.

Sudhir Paul

Opis

Antibodies are the instraments of immune defense and attack. They can bind small atomic arrays as well as large epitopes with high affinity. Antibody Engineering Protocols presents advanced protocols in the field of antibody engineering, reviews of basic principles and methodology, and a historical perspective on the development of cur rently held beliefs about antibody structure-function relationships. The topics cover analysis of antibody sequences, three-dimensional structure, delineation of antibody characteristics in polyclonal mixtures, phage display of natural and synthetic antibodies, and antibody catalysis. Ligand recognition by antibodies occurs primarily at a subset of amino acid residues located in the complementarity determining regions (CDRs) found in the light (L) and heavy (H) chain subunits. Specific antibodies are developed by immunization with molecules identical to or related in structure to the target ligand. The immune repertoire from nonimmunized individuals also contains pre-existing specificities that can be selected by screening libraries composed of hybridoma cells or phage particles displaying F domains or indi vidual variable domains of the light (VJ and heavy (V DEGREES DEGREES) chains. Ran dom or site-directed mutagenesis in vitro can be used to refine the pre-existing specificities or produce new specificities de novo. Another level at which new specificities may be generated in vitro is V DEGREES and V DEGREES domain-swapping and CDR-swapping. The former procedure embodies a variation of natural mechanisms of generating antibody diversity. The latter procedure produces new intramolecular CDR combinations not found in nature.

Spis treści

SEQHUNT: A Program to Screen Aligned Nucleotide and Amino Acid Sequences. Molecular Modeling of Antibody-Combining Sites. Structure and Properties of Human Immunoglobulin Light-Chain Dimers. Crystallographic and Chromatographic Methods for Study of Antibody Light Chains and Other Proteins. Detection of Human Variable Gene Family Expression at the Single-Cell Level. Purification of Reduced and Alkylated Antibody Subunits. Murine Monoclonal Antibody Development. Comparative Properties of Polyclonal and Monoclonal Antibodies. Evaluation of Antibody Clonality. Affinity Immunoblotting. Epitope and Idiotope Mapping Using Monoclonal Antibodies. Anti-Idiotypic Antibodies That Mimic Opioids. Catalytic Antibodies: Structure and Possible Applications. Preparation and Assay of Acetylcholinesterase Antibody. DNA Hydrolysis by Antibodies. Screening Strategies for Catalytic Antitransition-State Analog Antibodies. Expression of Chimeric Immunoglobulin Genes in Mammalian Cells. Single-Chain Anti-DNA Fv. Molecular Cloning of Antiground-State Proteolytic Antibody Fragments. Cloning and Bacterial Expression of an Esterolytic sFv. Site-Directed Mutagenesis of Antibody-Variable Regions. Synthetic Antibody Gene Libraries for In Vitro Affinity Maturation. Chaperonins in Phage Display of Antibody Fragments. Phage-Display Libraries of Murine and Human Antibody Fab Fragments. Selection of Human Immunoglobulin Light Chains from a Phage-Display Library. Purification of Antibody Light Chains by Metal Affinity and Protein L Chromatography. Rapid Purification of Recombinant Antibody Fragments for Catalysis Screening. Assay of Radiolabeled VIP Binding and Hydrolysis by Antibodies. Methods of Measuring Thyroglobulin and Peptide-Methylcoumarinamide Hydrolysis by Autoantibodies. Radiolabeling of Antibodies for Therapy and Diagnosis. Index.