Further probes into quantitative aspects of competitive binding assays: allowance for effects of antigen multivalency in immunoassays.
Effects of antigen multivalency on procedures for the analysis of immunoassays are examined on the basis of a theoretical expression developed in the context of quantitative affinity chromatography [Nichol, L. W., Ward, L. D., and Winzor, D. J. (1981) Biochemistry 20, 4856-4860] but which is also pertinent to antigen-antibody interactions that may be described in terms of a single intrinsic association constant. Quantitative relationships are generated which provide the basis for more rigorous logit-log analyses of radioimmunoassays in which the antigen is multivalent, and an additional, theoretically superior, linear transform of the basic expression is developed. Simulated binding data for a tetravalent antigen system are then used to demonstrate the curvilinearity of the conventional Scatchard plot for such a system despite the homogeneity of binding sites, and the application of the various linear transforms involving logarithmic functions. Of particular interest in that regard is the observation that the traditional logit-log analyses yield linear plots with the predicted slope of unity even though antigen univalence is an implicit assumption in their application. Results obtained in a solid-phase radioimmunoassay of triiodothyronine are then presented to provide, for that system at least, experimental justification of the above-mentioned assumption that the antibody-antigen interactions may be described in terms of a single intrinsic association constant. Finally, an enzyme-linked immunoassay of ferritin is used to illustrate the possibility that a linear Scatchard plot may be obtained with a multivalent antigen under conditions where steric factors restrict participation of an antigen molecule to a single interaction with immobilized antibody.
D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis*; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D005293:Ferritins / Q000032:analysis; D007118:Immunoassay; D007700:Kinetics; D008962:Models, Theoretical; D011863:Radioimmunoassay; D014284:Triiodothyronine / Q000032:analysis
D000941:Antigens; D014284:Triiodothyronine; D005293:Ferritins