The measurement of relative antibody affinity by ELISA using thiocyanate elution

Abstract

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-ε{lunate}-DNP-L-lysine as antigen. A significant correlation (P < 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. © 1988.