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Updated: Jul 28, 2026
Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)
Published on: November 29, 2014
Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance
Y Y Yu1, B J Van Wie, A R Koch
1Department of Chemical Engineering, Washington State University, Pullman, Washington 99164, USA.
Insights
This study validates a biosensor design using real-time analysis of biomolecular interactions. The biosensor detects cardiac troponin I (TnI) and nicotinic acetylcholine receptors (nAChRs) through competitive binding reactions.
Area of Science:
- Biochemistry
- Biosensor Technology
- Immunology
Background:
- Biosensors are crucial for detecting biomarkers.
- Real-time analysis of biomolecular interactions provides insights into sensor performance.
- Understanding competitive binding reactions is key to biosensor design.
Purpose of the Study:
- To qualitatively verify a biosensor design based on competitive binding reactions.
- To analyze real-time biospecific interactions involving cardiac troponin I (TnI) and nicotinic acetylcholine receptors (nAChRs).
- To assess the kinetic properties of various antibody-antigen and antibody-receptor complexes.
Main Methods:
- Utilized BIAcore for real-time measurement of biospecific interactions.
- Immobilized TnI via covalent amine coupling on a CM5 sensor chip.
- Immobilized nAChRs noncovalently on an HPA sensor chip via hydrophobic adsorption.
- Calculated kinetic rate constants and affinities from BIAcore sensorgrams.
Main Results:
- Demonstrated successful immobilization of TnI and nAChRs while maintaining epitope accessibility.
- Quantified kinetic rate constants and affinities for multiple complex formations.
- Identified dissociation rate constants of the order of 10(-2) s-1 for specific complexes, enabling competitive binding.
Conclusions:
- The biosensor design, relying on competitive binding, was qualitatively verified.
- The characterized kinetic properties support the potential for competitive analyte binding in sensing applications.
- This work provides a foundation for developing advanced biosensing systems for cardiac and neurological targets.
Abstract:
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.
