Interaction between antibodies and hapten-protein conjugates of different composition: theoretical predictions and experimental data.

Mathematical models of competitive ELISAs with labelled antibody and with labelled antigen taking into account bivalent interactions between antibodies and hapten-protein conjugates were developed and analyzed. It was shown that in the kinetic model of the immunochemical reaction the conjugate composition influenced the amplitude of detected signal but not ELISA sensitivity. In the equilibrium model decreased sensitivity correlated with bivalent complexes formation. The predictions were tested experimentally using 2,4-dichlorophenoxyacetic acid (2,4-D) and testosterone as haptens. It was confirmed that increasing of the hapten : protein ratio resulted in formation of bivalent complexes with antibodies. The equilibrium binding constants for these complexes were two orders of magnitude higher than for monovalent ones. Optimal conjugate compositions have been chosen for ELISA of these haptens.