ELISPOT ASSAY


A modification of the ELISA assay called the ELISPOT assay allows the quantitative determination of the number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody . In this approach, the plates are coated with the antigen (capture antigen) recognized by the antibody of interest or with the antibody (capture antibody) specific for the antigen whose production is being assayed. A suspension of the cell population under investigation is then added to the coated plates and incubated. The cells settle onto the surface of the plate, and secreted molecules reactive with the capture molecules are bound by the capture molecules in the vicinity of the secreting cells, producing a ring of antigen-antibody complexes around each cell that is producing the molecule of interest. The plate is then washed and an enzyme-linked antibody specific for the secreted antigen or specific for the species (e.g., goat anti-rabbit) of the secreted antibody is added and allowed to bind. Subsequent development of the assay by addition of a suitable chromogenic or chemiluminescence-producing substrate reveals the position of each antibody- or antigen-producing cell as a point of color or light.

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Fields of Application of ELISPOT Assay


As it has been reported by Tanguay and Killion, ELISPOT appears to be 200 times more sensitive than ELISA in detecting secreted cytokines . These authors have shown that it was below delectability level of ELISA to detect cytokines released by less than lo4 cells, whereas as many as 10-100 cells per well was sufficient for the detection of cytokine-releasing cells. Such a high sensitivity makes ELISPOT a technique of choice for the detection of spontaneous and antigen-induced secretion of cytokines (e.g., interferon [IFNI-y, tumor necrosis factor [TNFI-a, interleukin [ILI-2, IL-4) from peripheral blood lymphocytes . ELSIPOT is widely used for vaccine development , AIDS research , cancer research , infectious diseases monitoring , autoimmune disease studies , and allergy and transplantation research.

ELISPOT Assay Historic Overview


In 1983, Sedgewick and Holt published a paper in the Journal of Immunological Methods describing a novel technique for the enumeration of antibody-secreting cells. The new technique was built on the same solid-phase immunoenzymatic principles as the enzyme-linked  immunosorbent assay (ELISA): antigen was immobilized to a solid support (plastic dish) to bind antibodies released by cultured splenocytes. Later, in 1983, another article describing

a similar antibody detection technique was published in the same journal by Czerkinsky and colleagues , who coined the name for this assay “enzymelinked immunospot,” or ELISPOT. Later, the original ELISPOT technique was modified in that the solid phase was coated with antibodies (rather than the antigen) to capture antigens (for example, cytokines) released by cultured cells . As modified, reversed ELISPOT has become very popular and appears to be

used more frequently than its predecessor. Some researchers call it “reversed ELISPOT,” whereas most truncated this name to just “ELISPOT” .

E-BOOK: Handbook of ELISPOT