Size-exclusion HPLC analysis of epitopes.

Abstract A size-exclusion chromatography method based on competition between intact antibodies and antigen-binding fragments (Fabs) is used to evaluate relative spatial location of epitopes recognized by monoclonal antibodies. If epitopes are spatially distinct, the presence of a potentially competitive Fab results in formation of complexes of higher molecular weight than those formed by antigen and intact IgG. Complexes of lower molecular weight are formed if the epitopes overlap, are identical, or if, upon formation of the antigen-antibody complex, conformational change occurs that results in loss of epitope. Size-exclusion chromatography of the relevant mixtures reveals these shifts in molecular weight distribution and, therefore, provides an unambiguous determination of spatially independent epitopes. In addition, chromatographic analysis of competition between IgG and its enzymatically prepared Fab is used to evaluate the dissociation rate constant of the antigen-antibody reaction in re-equilibrating mixtures.