Measurement of affinity of viral monoclonal antibodies by ELISA titration of free antibody in equilibrium mixtures.

The binding affinity of a monoclonal antibody (Mab) to tobacco mosaic virus (TMV) was determined by measuring, in an enzyme-linked immunosorbent assay, the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. In antibody excess, univalent binding of Mabs was observed and the affinity constant was K = 3.2 +/- 0.4 10(8) l/mol; in antigen excess, bivalent antibody binding was observed and the antibody avidity was about 15 times higher. In antigen excess, it was imperative to correct experimental data for the presence of 0.55% inactive molecules in the immunopurified antibody preparation. Modelling studies suggest that in the case of antibodies of increasing affinity, it becomes increasingly important to correct for the presence of inactive antibody in the binding assay.