ELISPOT

T cell ELISPOT assayELISPOT test

The T cell ELISPOT assay is a simple and sensitive immunoassay for the detection of protein secreting T cells at the single cell level.
 
U-CyTech biosciences offers a wide range of human, monkey, mouse and rat T cell ELISPOT kits including kits for the detection of cytokine 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 rhesus macaques, pig-tailed macaques, cynomolgous monkeys and baboons. 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 ELISPOT assay is one of the most sensitive tests to monitor ex-vivo cellular immune responses at the single cell level. The assay can accurately detect secreted proteins, such as cytokines, released by T cells in response to antigen. The cell suspensions, used in the test, can originate from blood (PBMC), lymphoid, spleen, bone marrow or CNS tissue. 

Classical T cell monitoring assays (e.g. Mixed lymphocyte reaction [MLR] and Cytotoxic T lymphocyte [CTL] assays) measure CD4+ or CD8+ T cell mediated immune responses. Both MLR and CTL assays have their drawbacks including the use of radioactivity, low throughput screening, decreased sensitivity in cryopreserved specimens and technical burden. RT-PCR analysis, to measure T cell responses can also be used. However, this assay detects mRNA instead of secreted protein.

 The ELISPOT assay, not afflicted with these shortcomings, has proven to be more sensitive than an ELISA or intracellular cytokine staining.1-3 The high sensitivity is due to plate-bound antibodies that directly capture the secreted proteins released by the cell before they dilute in the cell culture medium, are taken up by cells via cell-surface receptors or are degraded by proteases. This property enables the detection of very low frequencies of cytokine secreting cells (1/300,000) and also offers the possibility of high throughput screening. 
 

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.4

  • 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.5

  • The ELISPOT assay can be of help in understanding how new viruses cause disease and early vaccine development research, such as with the SARS-CoV-2 virus in the COVID-19 pandemic.6,7

  • The ELISPOT assay can be used to analyze T cell responses to predict allograft rejection.8

  • 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.9

  • The ELISPOT assay can be used to determine the frequency of antigen-specific T cell responses in PBMC of vaccinated non-human primates10,11,12 and spleen cells of immunized rats13.

  • The ELISPOT assay is currently being used increasingly for the quantitative assessment of peptide reactive T lymphocytes from PBMC in infectious diseases14,15, in the course of vaccination trials aimed at the induction of tumor-specific T cells16,17, or in the assessment of immune-mediated pathogenesis of autoimmune diseases18.

  • The ELISPOT assay is able to monitor the stimulatory effects of candidate adjuvants on cellular immune responses to co-administered vaccine proteins.19,20

 

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 T cell ELISPOT procedure are illustrated in the ELISPOT Flow diagram.
 

U-CyTech ELISPOT products

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ELISPOT applied in researchFAQs ELISPOT assayManual databaseReference databaseT cell ELISPOT productsOrdering information

 

References

Click on the authors for the abstract of the below mentioned papers.
 
  1. Tanguay S and Killion JJ (1994). Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine Cytokine Res. 13: 259-63.
  2. Carter LL and  Swain SL (1997). Single cell analyses of cytokine production. Curr. Opin Immunol. 9: 177-82.
  3. Herold KC et al. (2009). Validity and reproducibility of measurement of islet autoreactivity by T-cell assays in subjects with early type 1 diabetes. Diabetes. 58: 2588-95.
  4. Schloot NC et al. (2003). 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.
  5. Van der Meide PH et al. (1998). 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.
  6. Zhao B et al. (2021). Alterations in Phenotypes and Responses of T Cells Within 6 Months of Recovery from COVID-19: A Cohort Study. Virol. Sin. 2021 Feb 09: Online ahead of print
  7. Feng L et al. (2020). An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nat Commun. 11: 4207.
  8. Van Besouw NM et al. (2019). The number of donor-specific IL-21 producing cells before and after transplantation predicts kidney graft rejection. Front Immunol. 10: 748.
  9. Gerrits JH et al. (2009). T-cell reactivity during tapering of immunosuppression to low-dose monotherapy prednisolone in HLA-identical living-related renal transplant recipients. Transplantation 87907-14.
  10. Vierboom MPM et al. (2021). Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination. Cell reports. Med. 2: 100185.
  11. Rollier CS et al. (2016). T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates. Gene Ther. 23: 753-759.
  12. Tsujimura Y et al. (2020). Vaccination with Intradermal Bacillus Calmette-Guérin Provides Robust Protection against Extrapulmonary Tuberculosis but Not Pulmonary Infection in Cynomolgus Macaques. J. Immunol. 205 (11): 3023-3036.
  13. Liu GX et al. (2009). Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats. Vaccine 2009 May 14;27: 2940-7. 
  14. Skowera A et al. (2005). Analysis of anthrax and plague biowarfare vaccine interactions with human monocyte-derived dendritic cells. J. Immunol. 175: 7235-43.
  15. Hamano T et al. (2007) A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production. J. Virol. 81:1528-33.
  16. Mirandola L et al. (2019). A novel method for efficient generation of antigen-specific effector T-cells using dendritic cells transduced with recombinant adeno-associated virus and p38 kinase blockade. J. Transliterates. Med. 17: 424.
  17. Li J et al. (2007) Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy. PloS One. 2:e1122.
  18. Martin S et al. (2001). Development of type 1 diabetes despite severe hereditary B-cell deficiency. N. Engl. J. Med. 345: 1036-40.
  19. Younis S et al. (2018). Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation. BMC Immunol. 19: 6.
  20. Bogers WM et al. (2015). Potent immune responses in rhesus macaques induced by nonviral delivery of a self-amplifying RNA vaccine expressing HIV type 1 envelope with a cationic nanoemulsion. J. Infect. Dis. 211: 947-55.