Assessment Of Heterogeneity in Antibody−Antigen Displacement Reactions

Abstract

The intrinsic binding characteristics of monoclonal antibodies are modified upon immobilization onto a solid-phase matrix. Factors such as the distribution in affinity must therefore be taken into consideration in order to predict the kinetics of antibody binding at solid-liquid interfaces. A mathematical analysis is presented herein that allows the assessment of heterogeneity in the affinity of monoclonal antibodies immobilized onto a solid support. This model is based on a modified version of the Sips distribution function adapted to the conditions of a solid-phase displacement assay in flow. An assay for trinitrotoluene (TNT) provides the data to evaluate the extent of heterogeneity introduced by immobilization of antibodies in a flow immunoassay. We determined the index of antibody heterogeneity on two solid supports, controlled-pore glass beads and agarose beads, coated with a monoclonal anti-TNT antibody at varying densities. The data confirm that the threshold for crossover from homogeneous to heterogeneous forms of the reaction isotherm is different in displacement reactions than in association-dissociation reactions. Our analysis shows that the measured displacement isotherm is consistent with a homogeneous or only moderately heterogenous distribution of relative affinities.