ELISPOT | U-CyTech

T cell ELISPOT assay

U-CyTech biosciences offers a wide range of human, monkey, mouse and rat cytokine ELISPOT kits including kits for the detection of cytokine, chemokine or granzyme producing cells such as GM-CSF, Granzyme B, IFN-γ, interleukin family members (IL-2, IL-4, IL-10, etc.) and TNF-α. These ELISPOT kits are available in different formats (2- or 5-plate kits) and can be supplied with different coloring agents (either for silver or enzymatic staining producing "black" spots or "red" spots, respectively).
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

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).
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).
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).
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).
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

Click on the authors for the abstract of the below mentioned acticles.

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).

   U-CyTech products used in this study:
   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

2. van der Meide P.H., Groenestein R.J., de Labie M.C., Heeney J., Pala P. and Slaoui M.
   Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus
   macaques.
   J. Med. Primatol. 24:271-81 (1995).

   U-CyTech products used in this study:
   Monkey IFN-γ ELISPOT kit
   Monkey IL-2 ELISPOT kit
   Monkey species: Macaca mulatta

3. van der Meide P.H., de Labie M.C., Ruuls S.R., Groenestein R.J., Botman C.A., Olsson T. and Dijkstra C.D.
   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).

U-CyTech products used in this study:
Rat IFN-γ antibody

4. Gerrits J.H., van de Wetering J., Weimar W., and van Besouw N.M.
   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).

   U-CyTech products used in this study:
   Human IFN-γ ELISPOT kit
   Human IL-10 ELISPOT kit
   Human Granzyme B ELISPOT kit

5. van der Meide P.H., Joosten A.M., Hermans P., Kloosterman T.C., Olsson T. and de Labie M.C.
   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).

U-CyTech products used in this study:
Rat IFN-γ ELISPOT antibody pair

6. Ahmad, G., Zhang, W., Torben, W., Damian, R. T., Wolf, R. F., White, G. L., Chavez-Suarez, M., Kennedy, R. C., and
   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).

   U-CyTech products used in this study:
   Monkey IFN-γ ELISPOT kit
   Monkey IL-4 ELISPOT kit
   Monkey species: Papio anubis

7. Allen T.M., Vogel T.U., Fuller D.H., Mothe B.R., Steffen S., Boyson J.E., Shipley T., Fuller J., Hanke T., Sette
   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).

   U-CyTech products used in this study:
   Monkey IFN-γ ELISPOT kit
   Monkey species: Macaca mulatta

8. Skowera A., de Jong E.C., Schuitemaker J.H., Allen J.S., Wessely S.C., Griffiths G., Kapsenberg M. and
   Peakman M.
   Analysis of anthrax and plague biowarfare vaccine interactions with human monocyte-derived dendritic cells.
   J. Immunol. 175:7235-43 (2005).

   U-CyTech products used in this study:
   Human IFN-γ ELISPOT kit
   Human IL-2 ELISPOT kit
   Human IL-4 ELISPOT kit
   Human IL-13 ELISPOT kit

9. Hamano T., Matsuo K., Hibi Y., Victoriano A.F., Takahashi N., Mabuchi Y., Soji T., Irie S., Sawanpanyalert P.,
   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).

U-CyTech products used in this study:
Human IFN-γ ELISPOT kit

10. Chiriva-Internati M., Yu Y., Mirandola L., Jenkins M.R., Chapman C., Cannon M., Cobos E., and Kast W.M.
Cancer testis antigen vaccination affords long-term protection in a murine model of ovarian cancer.
PLoS One 5:e10471 (2010).

U-CyTech products used in this study:
Mouse IFN-γ ELISPOT kit

11. Li J., Zeng X.H., Mo H.Y., Rolen U., Gao Y.F., Zhang X.S., Chen Q.Y., Zhang L., Zeng M.S., Li M.Z., Huang
   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).

U-CyTech products used in this study:
Human IFN-γ ELISPOT kit

12. Martin S., Wolf-Eichbaum D., Duinkerken G., Scherbaum W.A., Kolb H., Noordzij J.G., and Roep B.O.
Development of type 1 diabetes despite severe hereditary B-lymphocyte deficiency.
N. Engl. J. Med. 345:1036-40 (2001).

   U-CyTech products used in this study:
   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

13. Roep B.O. (2006).
      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

14. van der Meide P.H., Villinger F., Ansari A.A., Groenestein R.J., de Labie M.C., van den Hout Y.J., Koornstra
      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).

   U-CyTech products used in this study:
   Monkey IFN-γ ELISPOT kit
   Monkey IL-2 ELISPOT kit
   Monkey IL-4 ELISPOT kit
   Monkey IL-13 ELISPOT kit
   Monkey species: Macaca mulatta

15. Wu F., Yuan X.Y., Li J. and Chen Y.H.
   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