Antibody-antigen complex formation with immobilized immunoglobulins.
We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D000939:Epitopes / Q000276:immunology; D006735:Horseradish Peroxidase; D007124:Immunoenzyme Techniques; D007136:Immunoglobulins; D009419:Nerve Tissue Proteins / Q000276:immunology; D011374:Progesterone / Q000031:analogs & derivatives / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; C029152:G-substrate; D007136:Immunoglobulins; D009419:Nerve Tissue Proteins; D011374:Progesterone; D006735:Horseradish Peroxidase