The ELISpot Assay Background |
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Developed in 1983, the ELISpot assay represents the convergence of plate-based Enzyme Linked Immunosorbent Assays (ELISAs) with membrane-based Western blotting technologies, permitting detection of secreted analytes at the single cell level. Originally conceived for the enumeration of B cells secreting antigen-specific antibodies, the ELISpot has been adapted for many tasks, the most prominent being the quantification of antigen-specific cytokine responses (See image below). Since its initial development, the ELISpot assay has been commonly applied to inflammation and cytokine responses for a variety of diseases from infections to cancer.
(1) Coat membrane with capture antibody. Add immune cells and stimulate.
(2) Responding cells produce cytokines. The cytokine of interest binds to the capture Ab beneath the cell.
(3) Wash to remove cells. Add a second cytokine-specific biotinylated Ab which binds to the cytokine-Ab complex.
(4) Add streptavidin-enzyme conjugate.
(5) Add enzyme substrate and develop. Within a well, each responding cell will result in the development of one spot.