An analysis of antigen-antibody binding kinetics for biosensor applications utilized as a model system: influence of non-specific binding.

The influence of non-specific binding on the specific binding of antigen in solution to antibody immobilized on a biosensor surface is presented for first-, one and a half-, second-, and other order reactions occurring under external diffusion-limited conditions. Both single-step and dual-step binding of antigen to antibody is considered. For a half-order reaction the value of the ratio of non-specific binding to specific binding (alpha) does not affect the rate of specific binding since a single curve represents the binding curve for alpha = 0 to 0.5. An increase in the alpha value leads to a decrease in the rate of binding and in the amount of antigen bound specifically to the antibody on the surface for first-, one and a half-, and second-order reactions. Also, an increase in the reaction order increases the sensitivity of the specific binding to the alpha value. An increase in the antigen concentration in solution increases the amount and the rate of specific binding for first-, one and a half-, and second-order reactions. The introduction of non-specific binding leads to complexities in the specific binding of the antigen for the one and half- and second-order reactions as the antibody concentration on the surface is varied. When non-specific binding is present there is an optimum value of the antibody concentration on the surface that yields the maximum rate and amount of antigen specifically bound for one and a half- and second-order reactions. Though this optimum amount of antibody immobilized on the surface is the same for the one and a half- and second-order reactions, it is different for different alpha values. No such complexities are observed for first-order reactions. The inclusion of non-specific binding in the analysis provides a more realistic picture of the binding of the antigen in solution to the antibody immobilized on the surface. The figures that show the numerically calculated binding rates for different orders when non-specific binding is present, represent the most useful part of the analysis for readers who are interested in constructing biosensors, and should assist in the control and manipulation of these interactions at the surface. These curves can be used to interpret a far from ideal binding of antigen to an immobilized antibody surface or vice versa. More-or-less all of the analysis should also be applicable to analytical systems that would not be classified as biosensors (that is, immuno and receptor assays).