ASSESSMENT OF HETEROGENEITY IN ANTIBODY-ANTIGEN DISPLACEMENT-REACTIONS

The intrinsic binding characteristics of monoclonal antibodies are mod ified upon immobilization onto a solid-phase matrix, Factors such as t he 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 th e assessment of heterogeneity in the affinity of monoclonal antibodies immobilized onto a solid support, This model is based on a modified v ersion of the Sips distribution function adapted to the conditions of a solid-phase displacement assay in now. An assay for trinitrotoluene (TNT) provides the data to evaluate the extent of heterogeneity introd uced by immobilization of antibodies in a flow immunoassay. We determi ned the index of antibody heterogeneity on two solid supports, control led-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-d issociation reactions. Our analysis shows that the measured displaceme nt isotherm is consistent with a homogeneous or only moderately hetero genous distribution of relative affinities.