Wikipedia
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 (PBMCs).
The ELISPOT method was developed by Cecil Czerkinsky’s 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, PBMCs 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 “captured” in the area directly surrounding the secreting cell, before it has a chance to diffuse into the culture media, or to be degraded by proteases and bound by receptors on bystander cells. Subsequent detection steps visualize the immobilized cytokine as an ImmunoSpot; essentially the secretory footprint of the activated cell (see below).
This mechanism of capturing the secreting cytokine makes the ELISPOT method far more sensitive than assays that measure cytokine released into culture supernatants, for the above stated reasons. Cytokine Bead Arrays (CBA) and conventional ELISA assays can provide extremely useful information in certain contexts, but lack the sensitivity and accuracy of ELISPOT for the detection and enumeration of rare antigen-specific cells.
The practical limits of detection for ELISPOT are dependent generally on the number of cells seeded in an assay well. Typically 200,000 - 400,000 PBMCs will be used per well for an assay, but up to one million cells are commonly used for detection of rare events. ELISPOT is capable of detecting a single antigen positive cell within this population, giving it a theoretical low limit of detection of one in one million cells.