Ž . Journal of Immunological Methods 201 1997 189–206 Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system Jacob Piehler a, ) , Andreas Brecht a , Thomas Giersch b , Bertold Hock b , Gunter Gauglitz a ¨ a Institut fur Physikalische und Theoretische Chemie, Auf der Morgenstelle 8, D-72076 Tubingen, Germany ¨ ¨ b TU Munchen, Lehrstuhl fur Botanik, Weihenstephan, D-85350 Freising, Germany ¨ ¨ Received 31 May 1996; revised 8 October 1996; accepted 1 November 1996 Abstract We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference Ž . spectroscopy RIfS using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10 7 M y1 and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed. Keywords: Antibody affinity; Affinity constant; Label-free affinity probe; Monoclonal anti-s-triazine antibody 1. Introduction The strong and specific binding of target molecules to the binding sites of an antibody plays an important Ž role in several fields of analytical chemistry. The interaction between these target molecules termed ‘analytes’ ) Ž. Ž. Corresponding author. Tel.: q49- 0 7071-29-74667; Fax: q49- 0 7071-29-6910; e-mail: jacob.piehler@uni-tuebingen.de 0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0022-1759 96 00222-0