METHOD FOR DETERMINING THE AFFINITY OF MONOCLONAL-ANTIBODY USING NONCOMPETITIVE ELISA - A COMPUTER-PROGRAM

A simple and reliable method based upon law of mass action for calcula ting: affinity of a monoclonal antibody using: non-competitive ELISA, is described. In this method, the binding of an antibody (Ab) with an antigen (Ag) is measured by ELISA using serial dilutions of both antig en (coated on the plate) as well as antibody. When the OD measured aft er the antigen antibody interaction was plotted against the concentrat ion of Ab, added to the wells, a hyperbolic curve was obtained. The OD , at any point of the curve, was considered as a direct reflection of the amount of antibody bound to the antigen. The OD-100 denotes the oc cupancy of maximum no, of epitopes available on the antigen molecules, accessible to the antigen. The concentration of antibody (Ab, Ab') at corresponding levels of antigen concentration (Ag, Ag'), presents the value obtained at OD-50. The [Ag] and [Ag'] are not the true antigen concentrations but are the measurement of antigen density on the plate . The affinity constant K-aff was calculated by using the formula K-af f = (n - 1)/2(n[Ab'] - [Ab]), derived from law of mass action, where n = [Ag]/[Ag']. A computer program to calculate the affinity of antibod y to the antigen using method described in this manuscript has been de veloped and discussed.