Identifying Residues in Antigenic Determinants by Chemical Modification

Chemical modification of the side-chains of residues in protein antigens was one of the first methods developed to investigate epitopes. Together with proteolytic fragmentation, it played a major role in the pioneering efforts of Atassi and others to assign antigenic determinants on the surfaces of lysozyme and myoglobin (1 ,2 ). The principle of the method is that alteration of the structure of a key residue in an epitope by a chemical modification reagent will greatly change its reactivity with an antibody to that epitope. The steps in the procedure are modification of the protein antigen with the chosen reagent, removal of byproducts after the reaction, and immunoassay of the modified protein for the expression of the epitope. The chemical modification is usually performed in solution, but in a convenient variation described here, the antigen is modified when it is already adsorbed to an ELISA plate (3 ).