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

The interaction between polyclonal and monoclonal antibodies to steroid hor mones, immobilised on microtiter plates through an anti-gamma-globulin seco nd antibody, and monosubstituted horseradish peroxidase-based tracers was s tudied. Experimental binding constants, density of antibody binding sites a nd dissociation rate constants for the antibody-tracer system were measured as the increasing surface density of immobilised antisteroid antibodies. T he binding constant remains equal to 2x10(8) M-1, when the polyclonal bindi ng site density is lower than 3 fmol cm(-2). Then the binding constant incr eases up to about 12x10(8) M-1, when polyclonal binding sites reach maximum surface density (ca. 5 fmol cm(-2)). When the monoclonal binding site dens ity increases from ca. 1 to ca. 22 fmol cm(-2), the binding constant rises from ca. 3 to ca. 26x10(8) M-1; then the binding constant splits into two c omponents, a higher constant, K-1, that increases sharply up to ca. 90x10(8 ) M-1 at maximum binding site density (ca. 40 fmol cm(-2)) and a lower cons tant, K-2, that is always equal to ca. 3x10(8) M-1. The gradual binding con stant increase, observed in polyclonal and monoclonal antibodies, could be justified by the dissociation rate constant decrease of the tracer from imm obilised antibodies at increasing surface binding site density; the sharp b inding constant increase and the binding constant splitting could be due to co-operative interactions between the adjacent immobilised monoclonal anti body molecules and between the two binding sites of each antibody molecule. (C) 1999 Elsevier Science B.V. Ail rights reserved.