The enzyme-linked immunosorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell. The ELISpot Assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte, with the word analyte referring to any biological or chemical substance being identified or measured. The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence in order to analyze multiple analytes, meaning it can detect the secretion of more than one type of protein. History Cecil Czerkinsky first described ELISpot in 1983 as a new way to quantify the production of an antigen-specific immunoglobulin by hybridoma cells. In 1988, Czerkinsky developed an ELISA spot assay that quantified the secretion of a lymphokine by T cells. In the same year, dual-color ELISpot was combined with computer imaging for the first time, which allowed for the enumeration and analysis of spots. 1988 also marked the first use of membrane-bottomed plates for performing these assays. Mechanism of ELISpot Antibody coating: Throughout the ELISpot Assay technique, different substances are added to and washed away from wells. Wells are found on a laboratory plate with tiny dishes/bowls that can be filled with a substance to be examined; the amount of wells on a plate varies, but it usually ranges from 16-100. The first substance added to the wells are cytokine specific monoclonal antibodies. These antibodies coat the walls of the wells for future binding to cytokine. The monoclonal antibodies means that the antibody is produced from a single cell lineage, and is only able to bind to one protein epitope. Polyclonal antibodies, on the other hand, are capable of binding to multiple epitopes of the same protein. Cell incubation: The desired cells being observed and analyzed are added to the wells. Each well can have the presence or absence of stimuli that activate the secretion of cytokine in cells. During cell incubation, the cells are allowed to react to any present stimuli and secrete cytokine. There are many procedures and methods to follow to ensure proper cell handling. To make sure that cells are of high quality, cells in blood samples should be lightly agitated if stored for longer than 3 hours, the blood samples should be diluted in PBS (phosphate buffered saline) before being stored, and the blood samples should not