An analytical solution to the characterization of antigen–antibody interactions by kinetic exclusion assay

A simpler derivation of the basic expression for the dependence of fluorimetric response ratio (RAg/Ro) on free antigen concentration has demonstrated the universal invalidity of the analysis that is incorporated into the manufacturer’s software for determining immunoaffinities by kinetic exclusion assay, and traced the error to inadequate allowance for antibody bivalence in the solution phase of the assay. An analytical solution to the quantitative characterization of antigen–antibody interactions from the dependence of RAg/Ro on total antigen concentration is also described, thereby eliminating the necessity for the extensive simulative procedures employed in current determinations of dissociation constants by kinetic exclusion assay. In the illustrative application of this analytical approach to published results on the interaction between a metal chelate (cadmium–ethylenediaminetetraacetic acid, Cd–EDTA) and an elicited monoclonal antibody, the analytical processing of the data has been performed on a calculator. However, there is no need to replace the more sophisticated procedure that is incorporated into the Sapidyne software provided that programming changes are made to rectify the erroneous equation on which the simulative analysis is based.