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ELISPOT

What the assay tells you

The ELISPOT is used to detect very small amounts of cytokines secreted by T cells in culture. It depends on concentrating the cytokine molecules in small areas of a culture well by having them bind to anti-cytokine antibodies coating the plate, removing the cells, then detecting bound cytokine with enzyme-labeled anti-cytokine and a chromogenic substrate for the enzyme. The assay allows one to count the number of cytokine-secreting cells in each well.

What you need to do the assay

• T cell culture: cells and culture fluid and system to stimulate cytokine secretion. Remember, T cells need to bind specific peptide on infected cell syngeneic (self) Class I MHC (for CD8 T cells) or APC Class II MHC (for CD4 T cells). To avoid the difficultuies of culture, you could look for effector lymphocytes in the blood at the appropriate time after infection or immunization.
• Purified antibody to cytokine.
• Standard solutions (positive and negative controls).
• Microtiter dishes: plastic trays with small wells in which the assay is done.
• Wash fluid (buffer).
• Enzyme-labeled anti-cytokine and chromogenic substrate (colorless substrate that becomes colored due to enzyme activity.)

How the assay is done

• Coat the microtiter plates with specific monoclonal antibody to one epitope on the cytokine (see sandwich ELISA) and then protein to cover nonspecific binding sites on the plastic. Wash.
• Add T cells under conditions that stimulate them to secrete cytokines; they must bind specific peptide + self MHC to be activated. Cells will settle on the bottom of the microtiter plate. Incubate for several hours-days to collect cytokine on specific antibody; wash off cells and unbound tissue culture supernatant.
• Add enzyme-labeled monoclonal antibody to another epitope on the cytokine and incubate to bind; wash away unbound antibody.
• Add chromogenic (colorless) substrate for enzyme that will be converted into a colored product.

How to interpret the results

For each cell producing cytokine, a "spot" of color will be detected following substrate incubation. A larger spot will indicate a T cell producing more cytokine than a T cell giving a smaller spot.

Additional comments

This assay has the sensitivity and specificity advantages of the ELISA, plus some added sensitivity compared to running an ELISA on a larger volume of culture supernatant into which the cytokine has diffused and been diluted. Kits are now available (example from Bioscience Technology) that allow for the detection of two different cytokines simultaneously.

Useful web sites

Source of ELISA kits to human molecules:

Eurosciences (follow product link to elispot) http://www.euroclone.net/

Biosciences Technology http://www.biosciencetechnology.com/ShowPR_Print~PUBCODE~090~ACCT~9000007890~ISSUE~0506~RELTYPE~PR~ORIGRELTYPE~FP~PRODCODE~00000317~PRODLETT~A~CALLFROM~RELPGM~Menu~Similar.html

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