KINETIC-ANALYSIS OF IMMUNOINTERACTIONS WITH COVALENTLY IMMOBILIZED FATTY-ACID-BINDING PROTEIN USING A GRATING COUPLER SENSOR

Application of a grating coupler sensor (GCS) to the real time investi gation of the interaction kinetics of covalently immobilized recombina nt bovine heart-type fatty acid-binding protein (H-FABP) and correspon ding antibody is described. The immobilization of the antigen is perfo rmed by activating the matrix hydroxyl groups with p-toluenesulfonyl c hloride (TSC) and afterwards coupling the protein by reaction with its nucleophilic aminogroups. Covalent coupling via TSC permits reproduci ble measurements of immunointeractions on the same grating coupler sen sor chip and complete regeneration after each binding cycle with glyci ne-hydrochloride. We demonstrate the analysis of binding data obtained on a GCS by linearization as well as direct curve fitting using the i ntegrated rate equation for the determination of apparent rate and aff inity constants. With both analysis methods we studied H-FABP/monoclon al anti-H-FABP-antibody interactions and obtained an average apparent association rate constant k(a) = 4.2 x 10(3) M-1 s(-1), a dissociation rate constant of k(d) = 1.3 x 10(-4) s(-1) and an equilibrium constan t of K-D = 3 x 10(-8) M. (C) 1998 Published by Elsevier Science B.V. A ll rights reserved.