HI-CeFSpot: High-throughput Immune Cell FluoroSpot assay

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.