EQUILIBRIUM AND KINETIC-PARAMETERS FOR THE INTERACTION OF PEROXIDASE AND ITS CONJUGATES WITH SPECIFIC ANTIBODIES IN SOLUTION

The interactions of free and IgG- or cortisol-conjugated horseradish p eroxidase (HRP) with polyvalent antibodies against HRP (antiHRP) have been studied using a homogeneous enzyme immunoassay technique. Total c atalytic activities of free HRP or its conjugates and of their complex es, which are formed in time, with antiperoxidase antibodies (antiHRP) in o-phenylenediamine oxidation were measured. Time dependencies of t he initial rates of the peroxidase reaction linearized in the coordina tes {ln(upsilon - upsilon(infinity)); time}, {[antiHRP]/(upsilon - ups ilon(infinity)); [antiHRP](0)}, and {1/([antiHRP](0).k(1) + k(-1)); [a ntiHRP](0)} (where upsilon and upsilon(infinity) are rates at specifie d time and after reaching the equilibrium of the immune complex format ion, respectively) yielded the dissociation constants K-d for the immu ne complexes and the rate constants for their formation (kl) and disso ciation (k(-1)). The values of K-d found in this way agree reasonably with those determined from the classical Scatchard plot.