(Enzyme-linked ImmunoSPOT)

Evaluate cytokine production of individual cells

ELISPOT enables the analysis of activated or responding cells at the single cell level. ELISPOT are widely used for identification and enumeration of cytokine-producing cells. The method employs the sandwich assay approach of ELISA, with some modifications. The ELISPOT capture antibody is coated aseptically onto a PVDF membrane-backed microwell plate. Cells of interest are added at varying densities/well, along with antigen or mitogen, and plates are incubated at 37°C. Cytokines secreted by activated cells are captured locally by the coated antibody on the PVDF membrane. A biotin-conjugated detection antibody that recognizes a distinct epitope on the target cytokine is employed to detect the captured cytokine. This complex is visualized using avidin-HRP and a precipitating substrate. The colored end product (spot) reveals an individual cytokine-producing cell. Spots can be visualized manually (microscopy) or seen by using an automated reader to capture the microwell images to analyze spot number and size.

By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific response) is possible. Limits of detection with ELISPOT are below 1/100,000, rendering the assay uniquely useful for monitoring antigen-specific responses.

Advantages of using Ready-SET-Go! ELISPOT Sets

• Qualitative – visualize individual cytokine-producing cells by microscopy.
• Quantitative – enumerate activated, cytokine-secreting cells in 96-well plates, ideal for automation.
• Exquisite Sensitivity – greater than conventional ELISA, with limits of detection < 1/100,000 protein-secreting cells.

Human IL-17A ELISPOT

Human PBMCs cultured for 24 hrs (no mitogen).	Human PBMCs activated with PMA/Ionomycin for 24 hrs.