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Published on: May 2, 2025
High-throughput immunophenotyping by surface plasmon resonance imaging
Koichi Kato1, Toshinari Ishimuro, Yusuke Arima
1Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Insights
This study introduces surface plasmon resonance (SPR) imaging for quantitative cell binding detection on antibody arrays. This breakthrough enhances immunophenotyping throughput by enabling parallel and precise analysis of captured cells.
Area of Science:
- Biotechnology
- Immunology
- Analytical Chemistry
Background:
- Cell binding assays on antibody arrays enable rapid immunophenotyping.
- Current methods face throughput limitations due to challenges in parallel and quantitative cell detection.
Purpose of the Study:
- To develop and validate an imaging technique for quantitative cell detection on antibody arrays.
- To overcome the throughput limitations of current cell-based immunophenotyping methods.
Main Methods:
- Fabrication of antibody arrays via photopatterning of alkanethiol monolayers on gold-coated glass.
- Immobilization of antibodies onto distinct spots for parallel binding assays.
- Utilizing surface plasmon resonance (SPR) imaging to monitor refractive index changes caused by cell binding.
- Employing confocal laser scanning microscopy to analyze cell-antibody interactions.
Main Results:
- SPR imaging successfully detected intensified signals on specific antibody spots corresponding to cell capture.
- Observed SPR signals correlated with cell deformations resulting from multivalent antibody interactions.
- Quantitative monitoring of cell binding to multiple antibody spots from heterogeneous cell populations was achieved.
Conclusions:
- SPR imaging offers a novel, quantitative method for analyzing cell binding on antibody arrays.
- This technique significantly enhances the throughput and precision of immunophenotyping.
- The method holds potential for applications in diagnostics and drug discovery involving cell-surface interactions.
Abstract:
Cell binding assays on antibody arrays permit the rapid immunophenotyping of living cells. The throughput of the analysis, however, is still limited due to our inability to perform parallel and quantitative detection of cells captured on the array. To address this limitation, we employed here an imaging technique based on surface plasmon resonance (SPR). SPR has been frequently used to monitor capture of proteins on antibody microarrays, while few cases were reported for capture of cells. Antibody arrays were prepared through the photopatterning of an alkanethiol monolayer on a gold-evaporated glass plate and the subsequent immobilization of various antibodies onto 4-9 separate spots created by photopatterning. A glass slip was mounted onto the array with a thin spacer to construct a parallel-plate chamber. Leukemia cells were injected into the chamber to conduct a binding assay, while refractive index changes at the vicinity of the array surface were monitored by SPR imaging. We observed that SPR signals were intensified on specific antibody spots but not on nonspecific spots. Confocal laser scanning microscopy revealed that the observed SPR signals were attributed to cell deformations caused by multivalent interactions with immobilized antibody, which effectively elevated the refractive index of a medium phase within an evanescent field. This effect could be suitably utilized to monitor quantitatively cell binding to multiple spots from a heterogeneous cell population.
