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.
As noted above, the ELISPOT assays employ a technique very similar to the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal (preferred for greater specificity) or polyclonal capture antibody is coated aseptically onto a PVDF-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. Some antigens such as proteins may require to be previously loaded processed by antigen presenting cells.
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 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 streptavidin conjugated to an enzyme — horseradish peroxidase (HRP) or alkaline phosphatase (AP) — and a precipitating substrate (e.g., AEC, BCIP/NBT). The colored end product (a spot, usually red (for HRP) or a blackish blue (for AP)) 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.
The FluoroSpot assay is a modification of the ELISPOT assay and is based on using multiple fluorescent-labeled anti-cytokine antibodies which makes it possible to measure two cytokines in the same assay.
In 2011, in vitro tests based on detection of cell-mediated immunity for the diagnosis of tuberculosis became commercially available. These tests use the Mycobacterium tuberculosis (MTB)-specific antigens Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein 10 (CFP-10) peptides to stimulate M. tuberculosis-sensitized T-cells for the production of IFN-γ. ESAT-6 and CFP-10 antigens are specific for MTB and are produced from genomic area called Region of Difference 1 (RD1). Therefore, ELISPOT based detection of CME can provide valuable information about the diagnosis of tuberculosis. Most importantly, a recent study found that this test does not appear to have cross reactivity with leprosy even though L-ESAT, an M. leprae antigen is very homologous to the T-ESAT-6 used in this test. Therefore, this test can be even used in countries where leprosy is still endemic.
ELISPOT testing has been developed for detection of occult Lyme Disease infection.
Notes and references
- 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): 109–121. doi:10.1016/0022-1759(83)90308-3. PMID 6361139.
- Navarrete, MA (2015). "ELISpot and DC-ELISpot Assay to Measure Frequency of Antigen-Specific IFNγ-Secreting Cells.". Methods in molecular biology (Clifton, N.J.) 1318: 79–86. PMID 26160566.
- Shrestha R, Gyawali P, Yadav BK, Dahal S, Poudel B, Khanal M, Jha B, Sapkota B (2011). "In-vitro assessment of cell-mediated immunity by demonstrating effector-t cells for diagnosis of tuberculosis in Nepalese subjects.". Nepal Med Coll J. 13 (4): 275–8. PMID 23016479.
- JIn, Chenggang; Roen, Diana; Lehmann, Paul; Kellermann, Gottfried (2013). "An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi". Cells 2 (3): 607–620. doi:10.3390/cells2030607. PMC 3972671. PMID 24709800.