Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows: antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. 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 the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.