BINDING-KINETICS OF MONOCLONAL-ANTIBODY USING ANTIGEN-BETA-GALACTOSIDASE HYBRID PROTEIN - APPLICATION TO MEASUREMENT OF PEPTIDE ANTIGENICITY

A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluate d. To minimize conformational change of the antigen binding site of th e antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen conc entrations were directly obtained from the beta-galactosidase activity . This method can be used for analyses of the equilibrium dissociation constant (K-D), and the association (K-ass) and dissociation (K-diss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and th e peptide used are localized in the fluid-phase, the proper affinity c onstant (K-A) of the peptide can be estimated from the K-D value of th e antigen-beta-galactosidase-antibody interaction, and from the IC50 v alue of the peptide.