Kinetic analysis of immunointeractions with covalently immobilized fatty acid-binding protein using a grating coupler...

Application of a grating coupler sensor (GCS) to the real time investigation of the interaction kinetics of covalently immobilized recombinant bovine heart-type fatty acid-binding protein (H-FABP) and corresponding antibody is described. The immobilization of the antigen is performed by activating the matrix hydroxyl groups with p-toluenesulfonyl chloride (TSC) and afterwards coupling the protein by reaction with its nucleophilic aminogroups. Covalent coupling via TSC permits reproducible measurements of immunointeractions on the same grating coupler sensor chip and complete regeneration after each binding cycle with glycine-hydrochloride. We demonstrate the analysis of binding data obtained on a GCS by linearization as well as direct curve fitting using the integrated rate equation for the determination of apparent rate and affinity constants. With both analysis methods we studied H-FABP/monoclonal anti-H-FABP-antibody interactions and obtained an average apparent association rate constant ka = 4.2 X 10(3) M(-1) s(-1) a dissociation rate constant of kd=1.3 X 10(-4) s(-1) and an equilibrium constant of KD=3 X 10(-8) M.