The effect of different antibody affinities on ELISA absorbance and titer

Abstract

Twenty-one rabbit serum samples were assayed for IgG antibodies against 3-iodo-5-nitrophenyl-epsilon-amino-n-caproic acid (NIP-cap) by equilibrium dialysis and enzyme-linked immunosorbent assay (ELISA). The results of ELISA, expressed either as absorbances or titers, did not correlate to antibody concentration or average affinity measured by the equilibrium dialysis. Affinity distributions of the antibodies within each sample were determined by equilibrium dialysis in 30 antigen concentrations. Thus, it was possible to study the correlation of ELISA results with separate ranges of antibody affinities. ELISA absorbances measured at a low sample dilution (1:80) correlated with high affinity antibodies (affinity greater than 8 X 10(-6) M-1). Use of high antigen density increased this correlation. End-point titers obtained at a low cut-off absorbance level tended to correlate to a larger range of affinities (affinity greater than 6 X 10(4) M-1). ELISA measures antibodies of different affinities depending on how the results are expressed. This can be utilized in qualitative characterization of the measured antibodies.