ASSESSMENT OF AFFINITY CONSTANTS BY RAPID SOLID-PHASE DETECTION OF EQUILIBRIUM BINDING IN A FLOW SYSTEM

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid ph ase by a heterogeneous phase detection scheme. Equilibrium concentrati on of free antibody binding sites was probed kinetically by direct opt ical detection of specific binding to an immobilised analyte derivativ e. The additional binding signal due to dissociation of the analyte-an tibody complex during detection was minimised by the use of fast flow- through conditions. The concentration of free antibody binding sites w as titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least sq uare fit of a model function. The affinity of monoclonal triazine anti bodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of fr ee binding sites was carried out by reflectometric interference spectr oscopy (RIfS) using flow injection analysis. The capabilities of the m odel were investigated using different analyte-antibody pairs and vari ous antibody concentrations. Both bivalent IgG and monovalent Fab frag ments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10 (7) M(-1) and higher. For lower affinity constants significant deviati ons due to dissociation of the analyte-antibody complex during detecti on were observed.