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Updated: Jul 15, 2026
Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)
Published on: November 29, 2014
Measurement of antibody-membrane interactions by surface plasmon resonance
Alexander Klimka1, Mehmet K Tur, Michael Huhn
1Department of Pharmaceutical Product Development, Fraunhofer IME, Worringerweg 1, 52074 Aachen, Germany.
Abstract:
Due to problems of immobilizing functional tumor antigens in their natural conformation on surfaces for immunoassays, it is often difficult to evaluate the binding of antibodies derived from phage display libraries depleted and selected by panning on cell lines and living tumor cells. Performing cell membrane based ELISA methods does not reveal any up front kinetic binding information and depends on the performance of secondary antibodies and substrates. To overcome these limitations, we developed a new method to visualize direct antibody-cell membrane interactions by surface plasmon resonance using the Biacore 3000 and on-line signal subtraction on antigen-negative cell membrane vesicles. Conditions for the coating of cell membrane preparations to a carboxymethyl dextran hydrogel surface of a commercially available chip and the proof of concept for this application by the analysis of different formats of anti-CD30 and anti-carcinoembryonic antigen (CEA) antibodies interacting with coated membrane vesicles of CD30-positive/CEA-negative and CD30-negative and CEA-positive cell lines are described.
Insights
This study presents a novel surface plasmon resonance method for direct antibody-cell membrane interaction analysis. This technique overcomes limitations of existing immunoassays, enabling precise kinetic binding data for antibody discovery.
Area of Science:
- Biochemistry
- Immunology
- Biotechnology
Background:
- Evaluating antibody binding to cell surface antigens is crucial for cancer diagnostics and therapeutics.
- Traditional immunoassays like ELISA have limitations in providing direct kinetic binding data and maintaining antigen conformation.
Purpose of the Study:
- To develop and validate a new method for visualizing direct antibody-cell membrane interactions.
- To overcome limitations of current immunoassays for analyzing antibody binding to functional tumor antigens.
Main Methods:
- Utilized surface plasmon resonance (SPR) with a Biacore 3000 instrument.
- Developed a method for coating cell membrane preparations onto a carboxymethyl dextran hydrogel chip.
- Employed on-line signal subtraction using antigen-negative cell membrane vesicles for accurate measurements.
- Analyzed interactions of anti-CD30 and anti-carcinoembryonic antigen (CEA) antibodies with specific cell membrane vesicles.
Main Results:
- Successfully established conditions for coating cell membrane preparations onto SPR chips.
- Demonstrated proof of concept by analyzing antibody-antigen interactions with high specificity.
- Obtained direct kinetic binding information for antibody-cell membrane interactions.
Conclusions:
- The developed SPR method enables direct visualization and kinetic analysis of antibody-cell membrane interactions.
- This technique offers a significant advancement over existing methods for evaluating antibodies targeting cell surface antigens.
- The method is applicable for analyzing antibodies against various tumor antigens like CD30 and CEA.
