T cell ELISPOT assay
The antibodies in the monkey kits kits have been validated for detecting cytokine producing cells of various Old World monkeys including barbary macaques, lion-tailed macaques, pig-tailed macaques, Sulawesi macaques, Japanese macaques, cynomolgous monkeys, Langurs, baboons and mangabeys. ELISPOT kits for other monkey cytokines will follow, including kits for Marmoset (New World Monkey species) cytokines.
Additionally, human and monkey "ELISPOT" kits have been developed for the detection of two cytokines released by a single T cell with the use of fluorescent-labeled antibodies (the so-called FluoroSpot). FluoroSpot kits for the detection of multiple cytokines are under development.
Intended use of the T cell ELISPOT assay
The cytokine ELISPOT assay is designed to enumerate cytokine secreting cells in single cell suspensions of lymphoid tissue, CNS tissue, bone marrow or preparations of peripheral blood mononuclear cells (PBMC). The assay has the advantage of detecting only activated/memory T cells and the cytokine release can be detected at the single cell level, allowing direct determination of T cell frequencies. The high sensitivity and easy performance, allowing a direct enumeration of peptide-reactive T cells without prior in vitro expansion, makes the ELISPOT assay eminently well suited to monitor T cell responses.
Application
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The ELISPOT assay is an effective tool to enumerate antigen-specific T cells in the circulation of immunized humans and animals at much lower frequencies than possible with other currently available methods (1, 2).
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The ELISPOT assay has proven to be a sensitive and unique system to follow disease progression in human individuals or animals. Several studies have indicated that alterations in the frequency of cytokine producing cells in different compartments of the body adequately reflect changes in immune function (3).
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The ELISPOT assay can be used to determine effects of drugs, chemicals or other compounds on cytokine secretion in vitro, thereby providing data on their putative modulatory effects on immune function in vivo (4, 5).
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The ELISPOT assay can be used to determine the frequency of antigen-specific cytotoxic T cells (CTL) in fresh unstimulated peripheral blood lymphocytes from vaccinated non-human primates (6, 7).
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The ELISPOT assay is currently being used increasingly for the quantitative assessment of peptide reactive T lymphocytes from PBMC in infectious diseases (8, 9), in the course of vaccination trials aimed at the induction of tumor-specific T cells (10, 11), or in the assessment of immune-mediated pathogenesis of autoimmune diseases (12,13).
- The ELISPOT assay is able to monitor the stimulatory effects of candidate adjuvants on cellular immune responses to co-administered vaccine proteins (14, 15).
Brief description of the T cell ELISPOT procedure
Cells are incubated for a defined length of time in the wells of the ELISPOT plate precoated with a high-affinity monoclonal antibody to which the cytokine, produced during incubation, will bind. Subsequently, cells are lysed and debris is washed away. Areas in which the cytokine has been captured by the coating antibody are detected with a combination of biotinylated anti-cytokine detector antibodies and enzyme-labeled streptavidin or anti-biotin antibodies. The last step in the assay is the addition of a substrate yielding a colored zone ('spot'), which reveals the site of cytokine secretion.
The different steps of the ELISPOT procedure are illustrated in the ELISPOT Flow diagram.
U-CyTech ELISPOT products
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References ELISPOT products
1. Schloot N.C., Meierhoff G., Karlsson Faresjo M., Ott P., Putnam A., Lehmann P., Gottlieb P., Roep B.O.,
Peakman M. and Tree T.
Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells. Report of the third
immunology of diabetes society T-cell workshop.
J. Autoimmun. 21:365-76 (2003).
Human IFN-γ ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-5 ELISPOT kit
Human IL-10 ELISPOT kit
Human IL-13 ELISPOT kit
Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus
macaques.
J. Med. Primatol. 24:271-81 (1995).
Monkey IFN-γ ELISPOT kit
Monkey IL-2 ELISPOT kit
Monkey species: Macaca mulatta
Discontinuation of treatment with IFN-beta leads to exacerbation of experimental autoimmune encephalomyelitis in
Lewis rats. Rapid reversal of the antiproliferative activity of IFN-beta and excessive expansion of autoreactive T cells
as disease promoting mechanisms.
J. Neuroimmunol. 84:14-23 (1998).
T-cell reactivity during tapering of immunosuppression to low-dose monotherapy prednisolone in HLA-identical living-
related renal transplant recipients.
Transplantation 87:907-14 (2009).
Human IFN-γ ELISPOT kit
Human IL-10 ELISPOT kit
Human Granzyme B ELISPOT kit
Assessment of the inhibitory effect of immunosuppressive agents on rat T cell interferon-gamma production using an
ELISPOT assay.
J. Immunol. Methods 144:203-13 (1991).
Siddiqui, A. A.
Protective and antifecundity effects of Sm-p80-based DNA vaccine formulation against Schistosoma mansoni in a
nonhuman primate model.
Vaccine 27:2830-7 (2009).
Monkey IFN-γ ELISPOT kit
Monkey IL-4 ELISPOT kit
Monkey species: Papio anubis
A., Altman J.D., Moss B., McMichael A.J. and Watkins D.I.
Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus
macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.
J. Immunol. 164:4968-78 (2000).
Peakman M.
Analysis of anthrax and plague biowarfare vaccine interactions with human monocyte-derived dendritic cells.
J. Immunol. 175:7235-43 (2005).
Human IFN-γ ELISPOT kit
Human IL-2 ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-13 ELISPOT kit
Yanai H., Hara T., Yamazaki S., Yamamoto N., and Okamoto T.
A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion
production.
J. Virol. 81:1528-33 (2007).
Cancer testis antigen vaccination affords long-term protection in a murine model of ovarian cancer.
PLoS One 5:e10471 (2010).
W.L., Wang X.N., Zeng Y.X. and Masucci M.G.
Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor
immunotherapy.
PLoS One 2:e1122 (2007).
Development of type 1 diabetes despite severe hereditary B-lymphocyte deficiency.
N. Engl. J. Med. 345:1036-40 (2001).
Human IFN-γ ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-5 ELISPOT kit
Human IL-10 ELISPOT kit
Human IL-13 ELISPOT kit
T cell Assays.
In Rose N.R. and Mackay I.R. (ed.), p. 1023-30. The Autoimmune Diseases. Elsevier Inc., London.
U-CyTech products showed in this review:
Human IFN-γ ELISPOT
Human IL-10 ELISPOT
Human IL-13 ELISPOT
W.H., Bogers W.M. and Heeney J.L.
Stimulation of both humoral and cellular immune responses to HIV-1 gp120 by interleukin-12 in Rhesus macaques.
Vaccine 20:2296-302 (2002).
Monkey IFN-γ ELISPOT kit
Monkey IL-2 ELISPOT kit
Monkey IL-4 ELISPOT kit
Monkey IL-13 ELISPOT kit
Monkey species: Macaca mulatta
The co-administration of CpG-ODN influenced protective activity of influenza M2e vaccine.
Vaccine 27:4320-4 (2009).
U-CyTech products used in this study:
Mouse IFN-γ ELISPOT kit