Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay

Abstract

A reliable, convenient ELISA based method has been developed for measuring the dissociation rate constants of antigen/antibody complexes in solution. Its rationale is as follows: a solution containing the preformed antigen/antibody complex is diluted well below the equilibrium dissociation constant to initiate the dissociation and, at various times after the dilution, the amount of dissociated antibody contained in an aliquot is determined by a classical ELISA, using a brief incubation of the solution in antigen coated wells. To test the validity of this method, the dissociation rate constants for several antigen/antibody complexes were compared with those obtained by classical fluorescence based methods. The good agreement between both sets of data validates the ELISA procedure. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or antigen, and it can, in principle, be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.