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HI-CeFSpot: High-throughput Immune Cell FluoroSpot assay
Yogita Jethmalani1, Matthew S Sutton1, Robin Carroll1
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Methods (San Diego, Calif.)
|October 9, 2025
Summary
A new automated High-throughput Immune Cell FluoroSpot (HI-CeFSpot) assay improves precision and reproducibility for immune cell monitoring. This method enhances drug development, immunotherapy efficacy testing, and vaccine research.
Area of Science:
- Immunology
- Biotechnology
- Assay Development
Background:
- Immunospot assays like ELISpot and FluoroSpot are sensitive tools for immune cell profiling.
- FluoroSpot offers multiplexing capabilities with fewer cells and faster readouts than flow cytometry.
- Manual immunospot assays suffer from operator variability and low resolution.
Purpose of the Study:
- To develop and validate a High-throughput Immune Cell FluoroSpot (HI-CeFSpot) assay for automated immune cell monitoring.
- To assess the precision, robustness, and reproducibility of the automated HI-CeFSpot assay.
- To evaluate the HI-CeFSpot assay's performance using non-human primate immune cells stimulated with various agents.
Main Methods:
- Adaptation of FluoroSpot assay on a Biomek i7 liquid handler with automated plate washing and an IRIS 2 plate reader.
- Utilized non-human primate immune cells and screened against multiple stimuli to measure interferon gamma (IFN-γ) release.
- Evaluated assay performance by testing precision, robustness, and reproducibility across different cell types and densities.
Main Results:
- The HI-CeFSpot assay demonstrated high precision with <10% intra- and inter-plate variability and <15% inter-assay variability.
- The automated assay exhibited excellent reproducibility and robustness across multiple samples.
- Successful screening of immune cell responses to various stimuli was achieved with high throughput.
Conclusions:
- The developed HI-CeFSpot assay provides a high-throughput, precise, and reproducible platform for immune cell analysis.
- This automated assay is suitable for clinical trial endpoint testing, drug development, and vaccine research.
- HI-CeFSpot enables efficient and reliable immune cell monitoring and immunotherapy efficacy testing.
