FIGURE 8.3 Pharmacia BIACORE schematic representation. Inset shows typical response versus time curve (Byfield and Abuknesha, 1994).

Understandably, the mode of attachment or immobilization will significantly affect both nonspecific and specific binding of the antigen to the antibody or vice versa.

Disley et al. (1994) indicate the need to develop compatible interfaces to which the antibodies (or antigens) can be attached in a controlled and oriented manner, thereby assisting in either eliminating or minimizing nonspecific adsorption. The method suggested by these authors resulted in a reduction in the amount of nonspecific binding while simultaneously observing an increase in the specific binding of the polythiolated IgG interface for its target antigen. These authors suggest that the decrease in nonspecific effects was due to more mobile and hydrophilic surfaces, due to entropically driven protein monolayer repulsive interactions. More such studies are required to clearly delineate the method and the reason(s) why the nonspecific binding decreased.

Sadik and Wallace (1994) have emphasized that a significant cause of frustration is the fact that no surface is truly inert. This is derogatory as far as the development of stable and reversible biosensors is concerned. The authors indicate that "unruly" chemical processes occurring at biosensor surfaces will affect biosensor performance to different degrees. In essence, NSB occurs at surface sites where no antigen is adsorbed. Thus, the careful preparation of surfaces where the surface coverage is high would be helpful. In addition, free adsorption sites should be neutralized by inert molecules. Arwin and Lundstrom (1985) emphasize the importance of microorientation to minimize nonspecific adsorption wherein either the antigen or the antibody is immobilized on the surface. Here, due to the small number of determinants on the antibody molecules, it is important to orient the receptors on the surface toward the analyte in solution.

Immobilization of reagents for optical sensors is achieved in different ways. For example, immobilization can be achieved by adsorption on polymeric supports such as PTFE (Wyatt et al, 1987). Blair et al (1993) indicate that PTFE is an excellent surface for the immobilization of reagents since the reagent phase adsorbed on the PTFE is easily accessible to the analyte as compared to the adsorption of the reagent on the resin beads. Besides, the PTFE surface is inert, which eliminates or minimizes the nonspecific adsorption. Of course, it also makes the adsorption of the reagent more difficult.

Recently, there has been a significant effort to analyze and utilize surface-oriented affinity methods such as surface plasmon resonance (SPR). This technique should significantly assist in revealing the nature of the nonspecific and specific binding of the analyte in solution to the biosensor surface. Such a qualitative description that yields the details of the interactions of the molecules on the biosensor surface is of considerable utility. Pritchard et al. (1994) have emphasized that the major challenge faced during the design of a multianalyte immunosensor is the patterning of specific antibodies at discrete transducer sites and retaining the immunological activity. In addition, it is essential to minimize nonspecific effects.

Ekins and Chu (1994) have also discussed the development of multianalyte assays and their importance in microanalytical technology. Further development in multianalyte assays is required to increase their sensitivity and their reproducibility characteristics. These authors emphasize a miniaturized multianalyte system. Miniaturization would permit one to measure different analytes in very small samples. The extension of such multianalyte assay systems into conventional binding assay type techniques would permit their application into areas from which they were previously excluded due to cost and other complexities. Ekins and Chu are working the "microspot principle", which is basically that highly sensitive assays can be performed using much smaller amounts of antibody (confined to a "microspot") than previously thought necessary. Each microspot would then be used for different analytes. The authors do indicate that nonspecific binding will be a problem. Ways to minimize the effects of nonspecific binding include the selection of better supports, better antibodies or antibody fragments, and better instrumentation techniques. A time resolution of the fluorescent signal may be utilized to eliminate solid-support-generated backgrounds. Finally, Connolly (1994) indicates that the application of micro- and nano-fabrication techniques to biosensors should produce more oriented or specified arrays of antibody or antigen structures immoblized on the biosensor surface, besides minimizing nonspecific binding.

Marose et al. (1999) analyzed the use of optical systems for bioprocess monitoring. They indicate that although these systems exhibit potential for useful applications, there are some problems. These problems include the temperature dependence and that the optical sensors are subject to fouling. Of direct concern to us at present is that such systems need to be recalibrated often, which implies nonspecific binding. Since the possibility of continuous optical bioprocess monitoring is coming closer to reality, the issue of NSB needs to be addressed carefully. Very possibly, different methods of NSB minimization will be needed for different bioprocess applications.

Regarding another type of NSB, Williams and Addona (2000) have analyzed the successful integration of biospecific interaction analysis based on surface plasmon resonance combined with mass spectrometry. According to these authors, this technique combines the benefits of sensitive affinity capture and characterization of binding events with the ability to characterize interacting molecules. The amount of protein recoverable is in femtomoles. As expected, whenever extremely small amounts of analytes are involved, then NSB becomes increasingly important to maintain accuracy and precision. These authors indicate that due to NSB and dilution there may be a significant loss in the analyte due to the tubing and the recovery. In later versions of the instrument, NSB has been minimized by eliminating the recovery cup and minimizing the use of tubing. These improvements in minimizing the NSB and dilution effects (by using very small regenerant (for reverse flow elution)) permits a more accurate identification and characterization of the analyte molecules bound to the biosensor surface.

Ohlson et al. (2000) have analyzed the weak biological interactions that occur throughout biological systems either alone or in concert. They have designed a system that provides continuous monitoring using an on-line immunosensing device. One of the problems they encountered was nonspecific binding. This can be minimized by the selection of an appropriate reference system. Otherwise, nonspecific interactions may distort the interpretation of data.

Kortt et al. (1997) have indicated that nonspecific binding may be a cause for error in BIACORE binding measurements. These authors analyzed the interaction of monovalent forms of NC41 (an antiviral neuraminidase antibody) and 11-1G10 (an anti-idiotype antibody). The authors used this as a model system to demonstrate problems that may arise due to nonspecific amine coupling. Their results indicate that nonspecific immobilization (improper orientation) by one or more lysine residues close to or within the CDR2 region of the 11-1G10VH domain is presumably responsible for the decrease in interaction strength with NC41. This resulted in a reduction in the measured binding affinity. The authors emphasized that one should utilize site-specific immobilization strategies when accurate kinetic measurements are necessary.

Finally, Kubitschko et al. (1997) have attempted to enhance the sensitivity of optical immunosensors using nanoparticles. They indicate that due to low sensitivity many substances are not analyzable in serum. Nonspecific binding, along with the low molecular weight of the analytes, prevents an accurate quantitative determination of these analytes. They indicate that nonspecific binding makes a significant contribution to the signal and falsifies the result. This is especially true for smaller analytes, which cause only a small change in the refractive index on the sensor surface. The authors emphasize that for in vitro diagnostics small molecules, such as thyroid-stimulating hormone (TSH), need to be analyzed in serum so nonspecific binding needs to be minimized.

In this chapter, we will analyze theoretically and in some detail the influence of nonspecific binding and temporal model parameters on the specific binding of the antigen in solution to the antibody immobilized on the surface. The temporal nature of the specific binding parameters of the antigen in solution to the antibody immobilized on the surface, along with the inclusion of the nonspecific binding of the antigen in solution directly to the fiber-optic surface, is a more realistic approach to the actual situation. The analysis should provide fresh physical insights into first-, one-and-one-half-, second-, and other-order antigen-antibody reactions occurring on the fiber-optic biosensor surface under external diffusion-limited conditions.

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