Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance

Real-time biospecific interactions of immunogens, measured via BIAcore, were used to verify qualitatively a biosensor design which relies on analyte binding competition reactions to open cross-linked receptor channels. The complexes of importance are: (1) cardiac troponin I (TnI) and monoclonal mouse anti-TnI IgG mAb 265, (2) TnI and bispecific antibodies (BsAbs) which on one end recognize TnI while the other end recognizes nicotinic acetylcholine receptors (nAChRs), (3) nAChRs and rat anti-nAChR IgG mAb 148, (4) nAChRs and BsAbs, (5) nAChRs and Fab'148-TnI biopolymers, and (6) mAb 265 and Fab-TnI biopolymers. A commonly used sensor chip, CM5, was employed to immobilize TnI by covalent amine coupling, while bilayer membrane-associated protein, nAChR, was noncovalently sequestered on a HPA sensor chip via hydrophobic adsorption of membrane lipids. The epitopes of membrane-bound nAChRs were still available to immunogens after being immobilized. Kinetic rate constants and affinities of these systems were calculated from BIAcore sensorgrams. The order of magnitude for dissociation rate constants of the BsAb/TnI linker complex and biopolymer/mAb 265 complex is 10(-2) s-1, which provides an opportunity for competitive binding of free analyte in the sensing systems.