Affinity between immobilised monoclonal and polyclonal antibodies and steroid-enzyme tracers increases sharply at high surface density

Abstract

The interaction between polyclonal and monoclonal antibodies to steroid hormones, immobilised on microtiter plates through an anti-γ-globulin second antibody, and monosubstituted horseradish peroxidase-based tracers was studied. Experimental binding constants, density of antibody binding sites and dissociation rate constants for the antibody–tracer system were measured as the increasing surface density of immobilised antisteroid antibodies. The binding constant remains equal to 2×10 8 M −1, when the polyclonal binding site density is lower than 3 fmol cm −2. Then the binding constant increases up to about 12×10 8 M −1, when polyclonal binding sites reach maximum surface density (ca. 5 fmol cm −2). When the monoclonal binding site density increases from ca. 1 to ca. 22 fmol cm −2, the binding constant rises from ca. 3 to ca. 26×10 8 M −1; then the binding constant splits into two components, a higher constant, K 1, that increases sharply up to ca. 90×10 8 M −1 at maximum binding site density (ca. 40 fmol cm −2) and a lower constant, K 2, that is always equal to ca. 3×10 8 M −1. The gradual binding constant increase, observed in polyclonal and monoclonal antibodies, could be justified by the dissociation rate constant decrease of the tracer from immobilised antibodies at increasing surface binding site density; the sharp binding constant increase and the binding constant splitting could be due to co-operative interactions between the adjacent immobilised monoclonal antibody molecules and between the two binding sites of each antibody molecule.