Introduction
Enzyme-linked immunosorbent spot (ELISPOT) assays were originally developed to enumerate B cells secreting antigen-specific antibodies, but subsequently
the assay has been adapted for identification and enumeration of cytokine-producing cells at the single cell level. The method employs the sandwich
assay approach of the enzyme-linked immunosorbent assay (ELISA), with some variations. The ELISPOT capture antibody is coated aseptically onto a
polyvinylidene difluoride (PVDF)-backed microwell plate. The plate is blocked with serum proteins, cells of interest are plated out at varying densities, along
with antigen or mitogen, and plates are incubated at 37°C. Cytokine secreted by activated cells is captured locally by the coated antibody on the high surface
area PVDF membrane. The wells are washed to remove cells, debris, and media components. A second antibody (biotinylated) reactive with a distinct
epitope of the target cytokine is employed to detect the captured cytokine. The detected cytokine is then visualized using avidin-HRP, and a precipitating
substrate (e.g., AEC). The colored end product (spot) 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.
The ELISPOT assay enables analysis of activated or responding cells at the single cell level. By virtue of exquisite sensitivity of the ELISPOT assay, frequency
analysis of rare cell populations (e.g., antigen-specific responses) which were not possible by bulk assay methods are now possible. 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.
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Human IL-17 ELISPOT:
Left: Human PBMCs cultured for 24 hrs (no mitogen).
Right: Human PBMCs activated with PMA/Ionomycin for 24 hrs
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Human Granzyme B ELISPOT:
Left: Human PBMCs without mitogen cultured for 24 hrs.
Right: Human PBMCs activated with PMA/Ionomycin for 24 hrs.
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Reference Tables
Selected References
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Gebauer BS, et al. 2002. Evolution of the enzyme-linked immunosorbent spot assay for post-transplant alloreactivity as a potentially useful immune monitoring tool. Am. J. Transplant. 9: 857-866.
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Guerkov RE, et al. 2003. Detection of low-frequency antigen-specifi c IL-10-producing CD4(+) T cells via ELISPOT in PBMC: cognate vs. nonspecifi c production of the cytokine. J. Immunol. Methods. 279: 111-121.
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Kreher CR, et al. 2003. CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J. Immunol. Methods. 278: 79-93.
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Ott PA, et al. 2004. CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specifi c memory effector CD4 and CD8 cell populations in human diseases. J. Immunol. Methods. 285: 223-235.
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Smith JG, et al. 2001. Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin. Diag. Lab. Immunol. 8: 871-879.
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Shafer-Weaver K, et al. 2003. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity. J. Translational. Med. 1: 14.
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Rininsland F, et al. 2000. Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity. J. Immunol. Meth. 240:143-155.
