Cooperativity in the antibody binding to surface-adsorbed antigen.

The binding to surface-adsorbed antigen of monoclonal mouse IgG-antibodies (mAbs), with two different affinities to dinitrophenyl (DNP), was measured by a calibrated ELISA. The concentration-dependence of antibody binding to surface-bound antigen of different epitope densities was analysed using Scatchard plots. The dissociation of bound tritium-labelled antibodies was measured in the presence of unlabelled antibodies in the bulk. At low surface concentration of bound anti-DNP, both high-affinity mAb and low-affinity mAb show a positive cooperativity in the binding reaction to antigen of high epitope density. Using antigen of lower epitope densities, the positive cooperativity is more pronounced for low-affinity clones. At higher surface concentrations of bound anti-DNP, the Scatchard plots indicate a negative cooperativity of binding, which is also implied by the increased dissociation found in the presence of antibodies in solution. The study confirms previous findings that the binding of antibodies to surface-bound antigen not only depends on intrinsic antibody affinity measured in solution. Other factors, such as self-interaction, also affect the heterogeneous binding reaction.