Generating monoclonal antibody probes and techniques for characterizing and localizing reactivity to antigenic determinants

Abstract

This book: Epitope Mapping covers all the major methods for the identification and definition of epitopes. The Pepscan assay is used to define B cell epitopes and makes use of synthetic peptides but can only be used if the amino acid sequence is known. It can be adapted for the delineation of both helper T cells and cytotoxic T cells. The identification of combined B and T cell epitopes can also be achieved using synthetic peptides. There are other methodologies for analysing for cytotxic T cell epitopes such as the purification of antigens presented by MHC class I molecules and expression cloning. Site directed mutagenesis is also a powerful tool in epitope mapping and can be used to evaluate the role of single amino acids in immune complex formation. Protein footprinting makes use of monoclonal antibodies produced by hybridoma technology and relies on the fact that the epitope is protected from cleavage when bound as an antibody-antigen complex. It is only useful for small antigens. Other monoclonal antibody assays such as enzyme linked immunosorbent assay and haemaglutination and slot-blotting may also be used in epitope mapping. Random phage display libraries bring together the genetic and amino acid peptide sequence and can be screened with antibody and the resulting peptide DNA sequenced to confirm the amino acid sequence of a specific eptiope. Investigation of carbohydrates can also be useful to eptitope mapping as deglycosylation can lead to loss of antigenic activity. Epitopes are important to the pharmaceutical industry and wherever appropriate, pharmaceutical applications of the methods described are included. For each method there is a description of the technology, protocols, trouble-shooting, and advice on when to use the method. This book will therefore be invaluable to any researcher involved in epitope mapping.