ELISPOT Assays Visualize the Secretory Product of Individual Cells

ELISPOT assays are exceptionally sensitive since the product is captured directly around the secreting cell, before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements.

Chronically stimulated T cells downregulate their TCR zeta-chain while maintaining unaltered levels of TCR alpha/beta chain expression (reviewed in Baniyash, Michal. Nature Reviews|Immunology 4, p.675–687, 2004.). Although such T cells are functionally deficient, they will stain positive with tetramers. In contrast, ELISPOT assays will only detect relevant functional T cells.

Cytokine production can also be detected by intracytoplasmic staining and flow cytometry. Like ELISPOT, intracytoplasmic staining provides information on the actual frequency of antigen-specific T cells that produce a given cytokine. However, ELISPOT assays are orders of magnitude more sensitive in detecting low-frequency antigen-specific cells (see figure). Antigen-specific T cells frequently occur in frequencies lower than 1 in 10,000 in peripheral blood, placing them below the detection limit of flow cytometry but easily detectable via ELISPOT. Moreover, ELISPOT assays measure actually secreted molecules in pharmacologically untreated cells. This accounts for post-transcription regulation, thereby visualizing the biologically relevant products.

Unlike ELISPOT assays, cytokine bead arrays and ELISA assays do not provide frequency information and do not quantify the per-cell secretory activity. In contrast, ELISPOT assays can distinguish between few cells secreting large quantities of a molecule versus numerous cells secreting smaller quantities.

Unlike mRNA measurements that reflect cytokine expression at a single moment, ELISPOT assays capture the secretory product during the entire duration of the assay. This makes detection via ELISPOT insensitive to the fine kinetics of the cellular response. For the same reason, two-color ELISPOT assays are ideally suited for measuring the coexpression of different molecules by the same cell.

Cytokine and granzyme B ELISPOT assays have recently emerged as the most sensitive and robust technique for direct ex vivo monitoring of antigen-specific CD4 or CD8 T cells. By measuring the frequencies and cytokine signatures of individual cells, they establish the clonal size and effector class of the specific T cell pool.