The measurement of relative antibody affinity by ELISA using thiocyanate elution

A variation of the standard ELISA assay was used to determine the relative affinities of six murine monoclonal anti-dinitrophenol (DNP) antibodies for DNP-bovine serum albumin (DNP-BSA). The procedure involved exposing replicate wells containing antibody bound to antigen to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was utilized as a measure of affinity and an index (affinity index) representing a 50% reduction in initial absorbance was used to rank the anti-DNP antibodies in order of increasing affinity. For comparison, the affinity constants of the six monoclonals were determined by equilibrium dialysis with 3H-epsilon-DNP-L-lysine as antigen. A significant correlation (P less than 0.02) was observed between the ranking orders obtained using the two methods, thereby demonstrating the applicability of the elution technique for the measurement of relative antibody affinity.