The application of enzyme kinetics to the determination of dissociation constants for antigen-antibody interactions in solution.

The binding in ELISA procedures of a soluble antigen to a coated antibody in the presence of a second soluble antibody of different epitope specificity can formally be described by a mathematical model, identical to the one describing the random association between enzyme and substrate in a two substrate enzyme system. At low antigen levels, the concentration of both the coated and soluble (monoclonal) antibody, present in molar excess, can be varied and the resulting ternary complex can be detected directly or indirectly. Double reciprocal plots of the ELISA signal versus antibody concentrations yield straight intersecting lines. From the intersection, functional dissociation constants in solution can be determined. Alternatively, when the antigen is too small to accommodate two antibodies or when the binding affinities are low, the antibody of interest can both be insolubilized and at the same time be present in solution as a secondary antibody. In the presence of antigen, the coated and soluble antibody will compete for the same epitope, without formation of a ternary complex. On changing the concentration, both of coated and of soluble antibody, reciprocal plots of the bound antigen signal versus the concentration of competing antibody result in linear relationships yielding an intersection from which the functional antigen-antibody dissociation constant can be calculated.