MEASUREMENT OF THE DISSOCIATION RATE-CONSTANT OF ANTIGEN ANTIBODY COMPLEXES IN SOLUTION BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY/

A reliable, convenient ELISA based method has been developed for measu ring the dissociation rate constants of antigen/antibody complexes in solution. Its rationale is as follows: a solution containing the prefo rmed antigen/antibody complex is diluted well below the equilibrium di ssociation constant to initiate the dissociation and, at various times after the dilution, the amount of dissociated antibody contained in a n 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 requ ires no radioactive or fluorescent labelling of the antibody or antige n, and it can, in principle, be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitativel y one of the macromolecules.