The Enzyme-Linked ImmunoSpot (ELISpot) assay is a very sensitive immunoassay, allowing the detection of a secreted cytokine at the single cell level. With detection levels that can be as low as one cell in 100 000, the ELISpot is one of the most sensitive cellular assays available. Depending on the substance analyzed, the ELISpot assay is between 20 and 200 times more sensitive than a conventional ELISA. 

In fact, the ELISpot displays a similar sensitivity as a RT-PCR analysis but detects the secreted protein instead of th e mRNA. For cytokine analyses this is advantageous since many cytokines are translationally regulated. 

ELISpot analyses are also less impaired by binding proteins and protease activity since the analyte is bound to the capture antibody immediately after secretion. Due to its high sensitivity, the ELISpot has proven particularly useful when studying small populations of active cells such as those regularly found in specific immune responses.
As a first step in the ELISpot assay, cytokine-specific monoclonal antibodies are immobilized on a solid phase. We recommend the use of a 96-well microtiter plate with a polyvinyl-difluoride (PVDF) membrane. In the second step, the cells to be investigated are added to the wells in presence or absence of stimuli and incubated for a relevant time period to allow cytokine production. 

The secreted cytokine will bind to the capture antibodies in the vicinity of the producing cells and, after removal of the cells by washing, detection anti-cytokine antibodies are added. These antibodies are either directly conjugated with enzyme or biotinylated, in which case a third step with enzyme conjugated Streptavidin is required. Finally a substrate is added which will form a colored, insoluble precipitate when catalyzed by the enzyme. This way a visible spot corresponding to the location of the producing cell is formed. The spots can be counted under a dissection microscope or with an automated ELISpot reader and the frequency of positive cells registered. 

Mabtech also offers the ELISpotPRO kit, a ready to use one-step detection kit using precoated plates with capture antibody and an enzyme-conjugated detection antibody.