A theoretical analysis is presented for the influence of non-specific binding on the specific binding of antigen in solution to antibody immobilized on a biosensor surface 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 are considered. An increase in alpha (the ratio of non-specific binding to specific binding) and an increase in the forward binding rate coefficient leads to a decrease in the normalized antigen concentration near the surface, c(s)/c(0) and in the amount of antigen bound specifically to the antibody immobilized on the surface, Gamma(Ag)(s)/c(0) for a first-order reaction. The presence of non-specific binding complicates the influence of the forward binding rate coefficient, k(f)(s) on the Gamma(Ag)(s)/c(0) amount for the one and a half- and second-order reactions. Apparently, there is an optimum value of k(f)(s) that leads to the maximum amount of antigen that can be specifically bound to an antibody immobilized on the surface. The Gamma(Ag)(s)/c(0) value is rather sensitive to the order of reaction, and decreases considerably (by orders of magnitude) as one goes from first- to one and a half-, and to second-order reactions. Temporal forward binding rate coefficients more correctly represent the complexities and heterogeneities involved during the binding of antigen in solution to the antibody immobilized on the surface. The inclusion of non-specific binding along with temporal forward binding (increasing as well as decreasing) rate coefficients in the analysis provide a more realistic picture of the binding of the antigen in solution to the antibody immobilized on the surface. This should considerably assist in the control and manipulation of these interactions at the surface.
