Epitope Mapping Using pEX

Monoclonal and polyclonal antibodies are important tools in molecular and cell biology, allowing protein molecules to be purified (1 ), identified in immunoblots of gels (2 ) and sections of biological material (3 ), and probed for functionally active regions. Studies on the nature of antibody binding sites show that the molecular interaction between an antibody and its antigen is very specific and localized to a small patch on the surface of a molecule (4 ). For antibodies that inhibit molecular function, it is clearly of value to be able to map their binding site to a physical location on the polypeptide chain. Even antibodies that do not inhibit binding or catalysis of the protein can give useful information since the epitopes of antibodies are located on the surface of an antigen molecule (5 –8 ). Two classes of antibody binding site have been distinguished. In the first—“continuous” epitopes—synthetic peptides are capable of binding to the antibody (9 ), suggesting that a single loop of the polypeptide chain is predominantly responsible for the antibody binding. In the second—discontinuous epitopes—linear peptides do not bind to the antibody, but the important amino acids can be mapped by mutational analysis (10 ). These discontinuous epitopes can sometimes be mimicked by peptides that imitate the spacial orientation of the surface residues (11 ).