Antibody-antigen complex formation with immobilized immunoglobulins

Abstract

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.

Virus detection using filament‐coupled antibodies

“…A negative control anti-E segment was adjacent to increasing concentrations of anti-M13 spotting solution, and the filament was processed through capillary reaction chambers using parameters similar to previous virus detection experiments. As Figure 5 illustrates, the data shows an increase in signal intensity up to 600 mg/mL, after which the signal begins to drop, a trend that is consistent with the literature for antibodies immobilized on other polymers (Schramm and Paek, 1992;Schramm et al, 1993).…”

Characterization for Binding Complex Formation with Site‐Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

“…As the surface density increases in the random preparation, the antibodies may change their molecular conformation into a more accessible orientation via self-assembly [2]. However, the use of a large quantity of antibodies as the capture may cause unwanted problems, for example, an increase in non-specific binding of the enzyme-labeled detection antibody to the immobilized layer [18] or negatively influencing the system [2, 3]. The site-directly prepared fragments immobilized at a 10 times lower density than the random IgG gave approximately an identical sensitivity.…”

“…The complex formation of antigen with the immobilized antibody is affected mainly by three factors: method of immobilization, surface density of the antibody, and size of antigen [2]. In general, these alter not only the density of reactive binding sites on the surfaces, but also the binding affinity of the antigen to the immobilized antibody.…”

“…The colorimetric signal data obtained from the each analyte concentration were quantified as molar concentration using a linearized HRP standard curve [2]. The numerical values were then transformed into the analytical parameters for Scatchard plot analysis [13].…”

“…The solid-phase capture antibody, however, is very difficult to control at the level of the molecular state or orientation on the surfaces, which is a major factor that determines the degree of the complex formation [2]. Indeed, most immobilization methods (e.g., physical adsorption, covalent attachment, and binding via biotin-streptavidin linkage) [2, 3] have not yet been very successful in arranging or orientating the antibody molecules [4] in a manner that substantially improves the random nature of antibody immobilization inherent in the current methods. Random immobilization results in a low rate (e.g., 5 to 10%) of the active antibody density, that is, those that can participate in the binding reaction, which decreases even further when the substance to be analyzed is large in molecular size [2].…”