ANALYSIS OF ANTIGEN-ANTIBODY REACTIONS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A simple and convenient method for the analysis of antigen-antibody reactions using high-performance liquid chromatography is described. A mixture of human serum albumin and anti-albumin monoclonal antibody in a buffer was analyzed on a gel filtration column. After the free albumin and antibody were rapidly separated from the antibody-antigen immune complex, the three amounts in the mixture were directly calculated from the peak heights. The concentration of the antigen and antibody as well as other reaction conditions were easily varied by this method. The effects of the reaction time, the concentrations of albumin or antibodies, and the pH were examined. The reactivities of the antigen binding sites of immunoglobulin G, pepsin digested fragment F(ab')2 and reduced monomeric form Fab' were studied; the results suggest that the Fc region of IgG may participate in the antigen-antibody interaction at those binding sites. Epitope typing of monoclonal antibodies was also carried out with a combination of two antibodies. This represents a new immunochemical technique for characterizing antibodies and for the quantitative assay of protein antigens.