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Published on: March 10, 2017
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.
Insights
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.
Abstract:
Immunospot assays are known for high sensitivity and low material requirement. ELISpot and FluoroSpot assays have been frequently used in immune cell monitoring and profiling, specifically with T-cells and B-cells. FluoroSpot enables multiplexing, similar to flow cytometry, but has the added benefit of requiring fewer cells, higher throughput at screening immunogens, and a faster assay readout. Immunospot assays are generally performed manually and are prone to operator errors in plate handling, leading to overlapping spots with low resolution and high variability. Here, we describe the development of a High-throughput Immune Cell FluoroSpot (HI-CeFSpot) assay that has been adapted on the Biomek i7 liquid handler with labware storage, in conjunction with an automated plate washer with stacker, and the IRIS 2 plate reader from Mabtech attached to the Orbitor robotic arm. To develop the HI-CeFSpot assay, we used immune cells from non-human primates (NHPs) and screened them against various stimuli to test the release of interferon gamma (IFN-γ). We tested parameters such as precision, robustness, reproducibility, and compared two different cell types across various cell densities. We found that the HI-CeFSpot assay had intra- and inter-plate precision of <10 %, and inter-assay precision of <15 %. The assay showed high reproducibility and was robust across multiple samples. The HI-CeFSpot assay described here is a platform solution that can be used in clinical trial endpoint testing for drug development, immune cell monitoring, testing the efficacy of immunotherapy, and in vaccine research with high-throughput, high precision, reproducibility, and multiplexing.
