FIGURE 8.2 Procedure for the immobilization of the antibody using hydrophillic adsorption, hydrophobic adsorption, and amino silanization (Ahluwalia et al., 1991).
Other methods by which nonspecific binding may be minimized exist. For example, one may coat the surface with a hydrophilic material that exhibits a low interfacial energy (Scheller et al, 1991). The ratio of nonspecific to the total binding should be carefully examined, and if it is negligible (say, less than 5-10%, an arbitrary number) it should be explicitly stated (Sadana and Sii, 1992). Furthermore, the spatial distribution of antibody-coated colloidal gold particles over an antigen-coated surface by electron microscopy has been analyzed by Nygren (1988). This author noted that the nonspecific as well as the specific binding for this case exhibits a spatial distribution. Thus, nonspecific binding should be considered in the development of an appropriate model.
Okano et al (1992) have developed a heterogeneous immunoassay for attomole-level detection. These authors indicate that the sensitivity depends on both the reaction efficiency and the nonspecific adsorption of the microparticles on the microplate surface. More specifically, they indicate that there are two ways to increase the sensitivity of the assay. In the first method, the reaction equilibrium needs to be shifted between the microparticle-
conjugated antibody and antigen toward the binding state. The other method is to minimize the nonspecific adsorption. Often, these are at cross-purposes: One may increase the number of surface antibodies, which would enhance the reactivity of the microparticle-conjugated antibody, but overloading the antibodies on the surface increases the nonspecific adsorption. Okano et al. also indicate that homogeneity of the antibody on the detection surface plays an important role in the sensitivity. One must take care to ensure that the capture antibody is distributed uniformly on the microplate surface. A certain amount of capture antibody is essential, otherwise this uniformity is not maintained. The authors added bovine serum albumin, which increased the total amount of protein and helped the uniformity of the capture antibody, thus assisting in minimizing the nonspecific adsorption. In essence, the bovine serum acts as a protein matrix, which helps promote the uniform immobilization. For example, the authors indicate that adding 25 ¡xg of bovine serum albumin to 1 fig of capture antibody could lower the nonspecific adsorption of microparticles to one-fourth of that without serum albumin.
Byfield and Abuknesha (1994) indicate that different types of proteins, ions, or small organic molecules can interact with the surface in a nonspecific manner. This, as expected, causes a major reduction in the signal-to-noise ratio for antibody-antigen measurements (as compared to when only pure antibody or pure antigen is utilized). These authors indicate that nonspecific adsorption is minimized in the hydrogel matrix developed by Pharmacia based on carboxy-methylated dextran. This matrix is linked to the gold surface of a surface plasmon resonance biosensor chip by a linker layer of a hydroxyalkyl thiol. Nonspecific adsorption is reduced by the hydrophilic nature of the dextran matrix. Biological binding reactions are promoted due to the dominance of the electrostatic forces. This prevents nonspecific interactions, which are hydrophobic in nature. As an additional benefit, the physical barrier provided by the dextran matrix prevents other (undesirable) components from reaching the reaction interface, and the specific antibody-antigen reaction is optimized. See Fig. 8.3.
Nonspecific binding may occur directly on the biosensor surface area where no antibody is present. Also, during the immobilization procedure on the biosensor surface, nonspecific binding of the antibody may occur. Often, this immobilization procedure can lead to heterogeneities on the biosensor surface. These must be taken into account in the analysis of specific binding by an appropriate procedure. Ideally, of course, it is better to minimize or, if possible, eliminate nonspecific adsorption. Collison et al. (1994) have emphasized that although many biomolecule immobilization strategies have been developed—including entrapment, chemical cross-linking, electropoly-merization, and covalent attachment—they often lack the reproducibility and reliability required for the fabrication of biosensors on a commercial scale.
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