Quantitation of reversible binding by particle counting: hapten-antibody interaction as a model system

With a view toward developing a general method for measuring intrinsic equilibrium constants for the reversible interactions between two ligands, we used an antibody-hapten model system [2,4-dinitrophenyl (DNP) hapten and anti-DNP antibody] to explore an approach based on particle counting of uniform polystyrene spheres to which the hapten is coupled covalently. This approach was made possible by an optical pulse particle size analyzer that accurately counts individual sphere clusters and quantitates with high precision specific aggregation of spheres crosslinked by antibody. The reduction in crosslinking that results from competition for antibody binding sites between a soluble DNP ligand and immobilized DNP groups on the spheres provides the basis for measuring the intrinsic equilibrium constant for the soluble ligand-antibody interaction. The binding constants measured in this way for several DNP ligands and an anti-DNP antibody (2A1) agreed with the values obtained by conventional methods. The range of intrinsic equilibrium constants that can be determined by particle counting is likely to be exceptionally wide and a value as low as 10(3) liters/mol has been measured. And since all soluble antigens, regardless of their mass, acquire the same ability to scatter light as a result of their immobilization on the much larger uniform spheres (0.36 microns), the approach described here should be applicable to virtually any molecularly dispersed antigen and its monoclonal antibody.