Epitope Mapping of Protein Antigens by Competition ELISA

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen which are in direct contact with the antibody-binding site. For immunoassay purposes, however, it is often more important to know whether one antibody interferes with the binding of a second antibody against the same antigen or whether both antibodies can bind without mutual competition. Antibodies in the latter category must recognize different epitopes and are useful for two-site assays in which one antibody is used to capture the antigen from a complex mixture (e.g., plasma or tissue extracts), while a second is used to detect the captured antigen. Antibodies that do compete with each other probably recognize the same epitope or a very near neighbor (), but the possibility that they bind to widely separated epitopes and compete by altering the conformation of the antigen must always be considered. Epitope mapping is usually performed with monoclonal antibodies (MAb), since polyclonal antisera are usually directed against a number of different epitopes and results could be difficult to interpret.