Specificity considerations in cooperative immunoassays.
Mixtures of some monoclonal antibodies form circular antibody-antigen complexes, which facilitates their ability to bind antigen. This effect forms the basis of a potentially very sensitive assay procedure, the cooperative immunoassay (CIA). Unlike other immunoassays, in which binding can be characterized by a simple "binding constant," the binding of antigen by two antibodies in a CIA depends on several binding parameters, including the affinity of each antibody for antigen as well as the tendency of the reactants to form a circular complex. The ability of the CIA to distinguish between two similar molecules depends on the relative affinity of the antibodies for each antigen and on the ability of the antigens to participate in forming a circular complex. To study the binding of antibody mixtures to cross-reacting antigens, we devised a mathematical model to account for all possible antibody-antigen complexes, including those composed of circular complexes; however, we limited this model to the case in which one antibody was adsorbed to a solid phase. We illustrate here both theoretically and experimentally that a mixture of two antibodies in a CIA may have increased or decreased specificity, if circular complexes containing one or two molecules of cross-reacting antigen are formed. We discuss simple practical considerations that can help optimize specificity and sensitivity of solid-phase assays involving two monoclonal antibodies.
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D007118:Immunoassay / Q000379:methods*; D008433:Mathematics; D008962:Models, Theoretical
D000911:Antibodies, Monoclonal