Determination of Affinity Constant from Microfluidic Binding Assay

Abstract

We present a method of determining the affinity constant of a receptor-ligand pair using equilibrium binding in a microfluidic channel. This technique involves the immobilization of the receptor to the microchannel surface and perfusion of the ligand at a fixed flow rate. The ligand concentration in the eluent is monitored in real time. The break-through time of the ligand is then determined and compared with that of an appropriate, non-binding, reference molecule. The difference in break-through time can then be used in a mathematical model we have developed to determine the affinity constant of the receptor-ligand pair. Using this method, we have determined the equilibrium dissociation constant, KD, of a rat anti-insulin immunoglobulin and a fluorescein-conjugated human insulin. Fluorescein-conjugated aprotinin was used as the non-binding reference molecule. Our experiments yielded a KD value of 0.76 μM for this anti-insulin IgG and insulin-FITC pair. © International Federation of Medical and Biological Engineering 2009.