ELISPOT

The Enzyme-linked immunosorbent spot (ELISPOT) is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983.^[1]

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The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product 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 (type of immune protein) and quantitative (number of responding cells) information.

By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part due to the fact that the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering the assay uniquely useful for monitoring antigen-specific responses, applicable to a wide range of areas of immunology research, including cancer, transplantation, infectious disease, and vaccine development. The assay has gained a recent increase in popularity, especially as a surrogate measure for CTL responses, in large part because it is both reliable and highly sensitive.

While ELISPOT assay techniques have existed for more than two decades now advancements are still being made in the assay. Modern ELISPOT analysis is typically performed using ELISPOT readers, which employ computer vision techniques to enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection.

Procedure

As noted above, the ELISPOT assays employ a technique very similar to the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal capture antibody is coated aseptically onto a PVDF (polyvinylidene fluoride) -backed microplate. These antibodies are chosen for their specificity for the analyte in question. The plate is blocked, usually with a serum protein that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities, along with antigen or mitogen, and then placed in a humidified 37°C CO₂ incubator for a specified period of time.

Cytokine (or other cell product of interest) secreted by activated cells is captured locally by the coated antibody on the high surface area PVDF membrane. After washing the wells to remove cells, debris, and media components. A biotinylated polyclonal antibody specific for the chosen analyte is added to the wells. This antibody is reactive with a distinct epitope of the target cytokine and thus is employed to detect the captured cytokine. Following a wash to remove any unbound biotinylated antibody, the detected cytokine is then visualized using an avidin-HRP, and a precipitating substrate (e.g., AEC, BCIP/NBT). The colored end product (a spot, usually a blackish blue) typically represents an individual cytokine-producing cell. The spots can be counted manually (e.g., with a dissecting microscope) or using an automated reader to capture the microwell images and to analyze spot number and size.

References

1. ^ Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A (1983). "A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells". J Immunol Methods 65 (1-2): 109-21. PMID 6361139.