ELISPOT

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ELISPOT The ELISPOT Assay

Dr. Cecil Czerkinsky, D.D.M., Ph.D. invented the Enzyme-linked immunosorbent spot (ELISPOT) assay in 1983, a method for monitoring immune responses in humans and animals, which is now used world-wide.

The ELISPOT assay is based on, and was developed from a modified version os the ELISA immunoassay. While ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, the assay has subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate cellular concentrations the ELISPOT assays allows one to visualize the secretory product of activated or responding individual cells. Each spot developed 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.

Moreover, ELISPOT assays measure actually secreted molecules in pharmacologically untreated cells. This accounts for post-transcription regulation, thereby visualizing the biologically relevant products.

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 exceptionally sensitivity is in part due to the act that the product is captured directly 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.

1 Procedure
2 Special Notes
3 References
4 External links

[edit] 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 difluoride) -backed microplate. These antibodies are chosen for their specificity for the analyte in question. The plate is blocked, usually with a serum proteins that is non-reactive with any of the 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.

[edit] Special Notes

Chronically stimulated T cells down-regulate their TCR zeta-chain while maintaining unaltered levels of TCR alpha/beta chain expression. Although such T cells are functionally deficient, they will stain positive with tetramers. In contrast, ELISPOT assays will only detect relevant functional T cells.

Cytokine production can also be detected by intracytoplasmic staining and flow cytometry. Like ELISPOT, intracytoplasmic staining provides information on the actual frequency of antigen-specific T cells that produce a given cytokine. However, ELISPOT assays are orders of magnitude more sensitive in detecting low-frequency antigen-specific cells. Antigen-specific T cells frequently occur in frequencies lower than 1 in 10,000 in peripheral blood, placing them below the detection limit of flow cytometry but easily detectable via ELISPOT.

Unlike ELISPOT assays, cytokine bead arrays and ELISA assays do not provide frequency information and do not quantify the per-cell secretory activity. In contrast, ELISPOT assays can distinguish between few cells secreting large quantities of a molecule versus numerous cells secreting smaller quantities.

Unlike mRNA measurements that reflect cytokine expression at a single moment, ELISPOT assays capture the secretory product during the entire duration of the assay. This makes detection via ELISPOT insensitive to the fine kinetics of the cellular response. For the same reason, two-color ELISPOT assays are ideally suited for measuring the coexpression of different molecules by the same cell. Cytokine and granzyme B ELISPOT assays have recently emerged as the most sensitive and robust technique for direct ex vivo monitoring of antigen-specific CD4 or CD8 T cells. By measuring the frequencies and cytokine signatures of individual cells, they establish the clonal size and effector class of the specific T cell pool.

[edit] References

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