Estimation of solid phase affinity constants using resistive-pulses from functionalized nanoparticles.

This paper describes a method for estimating the solid phase affinity constant of antibodies by using resistive-pulse (Coulter counting) data from spherical nanoparticles that expose antigens. We developed this technique by analyzing data published recently by Saleh, O.A., Sohn, L.L., 2003a. Proc. Natl. Acad. Sci. U.S.A. 100, 820-824. These authors used resistive-pulse sensing to detect an increase in the diameter of streptavidin-functionalized colloids due to the binding of monoclonal anti-streptavidin antibodies. Based on further analysis of their data, we were able to determine the number of antibodies bound to the colloids at various antibody concentrations. This information made it possible to estimate the solid phase affinity constant of the interaction by fitting the data with binding isotherms that describe the binding equilibrium between antibody and antigen. We calculated a value of 2.6x10(8)+/-0.8x10(8) M-1 which is in agreement with the specifications of the supplier of the antibody.