ELISpot Assay

The Enzyme‑Linked ImmunoSpot (ELISpot) assay is a highly sensitive cellular immunoassay used to quantify antigen‑specific immune responses at the single‑cell level and is widely applied in immunogenicity assessments for vaccines and immunotherapies. The assay measures the frequency of cytokine‑secreting cells following antigen‑specific stimulation, providing functional information on cellular immune responses. Peripheral blood mononuclear cells (PBMCs) are incubated with the test antigen on membranes coated with capture antibodies specific for the cytokine of interest. Cytokines secreted by activated T-cells are captured locally and subsequently detected using enzyme‑conjugated secondary antibodies, resulting in the formation of discrete spots corresponding to individual cytokine‑secreting cells. Spot counts are directly proportional to the magnitude of the antigen‑specific cellular response.

ELISpot is valued for its high sensitivity, robustness, and suitability for high‑throughput analysis, enabling the detection of low‑frequency antigen‑specific cells. However, the assay provides limited phenotypic characterization and typically allows assessment of a restricted number of parameters simultaneously. The assay, which is routinely used and validated in VisMederi (Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium.; Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells. ), commonly evaluates cytokines like interferon‑gamma (IFN‑γ) and interleukin‑4 (IL‑4), often assessed in dual‑color ELISpot formats to characterize T‑helper cell responses.