the enzyme-linked immunospot (elispot) assay is a widely used method for monitoring cellular immune responses in humans and other animals, and has found clinical applications in the diagnosis of tuberculosis and the monitoring of graft tolerance or rejection in transplant patients. the elispot technique has proven to be among the most useful means available for monitoring  cell-mediated immunity, due to its sensitive and accurate detection of rare antigen-specific t cells (or b cells) and its ability to visualize single positive cells within a population of  peripheral blood mononuclear cells (pbmc). the elispot method was developed by cecil czerkinskyâs group in gothenburg, sweden in 1983,  for the purpose of detecting antigen-specific antibody secreting cells (asc) in a b cell elispot assay, which was a modification of a traditional sandwich  elisa immunoassay. the elispot assay has since been more widely adopted for the identification and enumeration of cytokine-producing cells at the single cell level, but is still used for detection of asc. simply put, at appropriate conditions the elispot assay allows visualization of the secretory product(s) of individual activated or responding cells. each spot that develops in the assay represents a single reactive cell. thus, the elispot assay provides both qualitative (regarding the specific cytokine or other secreted immune molecule) and quantitative (the frequency of responding cells within the test population) information. due to the exquisite sensitivity of elispot assays, analyses of the frequencies of rare antigen-specific cells within a test population, which had been impossible to perform prior to its development, have now become relatively simple. this exceptional sensitivity derives from the mechanics of the assay method itself. in an elispot assay, the membrane surfaces in a 96-well  pvdf-membrane microtiter plate are coated with capture antibody that binds a specific epitope of the cytokine being assayed. during the cell incubation and stimulation step, pbmc are seeded into the wells of the plate along with the antigen, and form a monolayer on the membrane surface of the well. as the antigen-specific cells are activated, they release the cytokine, which is captured directly on the membrane surface by the immobilized antibody. the cytokine is thus âcapturedâ in the area directly surrounding the secreting cell, before it has a chance to diffuse into the culture supernatant, or to be degraded by proteases and bound by rece
Immunologic_tests