Detection of cytokines secreting cells using the T cell ELISPOT assay

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 IFN-γ, interleukins (IL-2, IL-4, IL-10, IL-17A, etc.) GM-CSF, Granzyme B and TNF-α. These ELISPOT kits are available in different formats (2- or 5-plate kits) and supplied with enzymatic coloring agents (producing red spots).
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. Our ELISPOT kits for New World monkey have been validated for the detection of marmoset 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).


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, interact with cell-surface receptors on cells or are degraded by proteases. This property enables the detection of very low frequencies of cytokine secreting cells (1/100,000) and also offers the possibility of high throughput screening. 


  • 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.4

  • The ELISPOT assay can be helpful in understanding the development of disease in cases of new viruses cause disease and in early vaccine development research, such as with the SARS-CoV-2 virus in the COVID-19 pandemic.5,6

  • The ELISPOT assay can be used to analyze T cell responses to predict allograft rejection.7

  • The ELISPOT assay can be used to determine the frequency of antigen-specific T cell responses in PBMC of vaccinated non-human primates8,9 and spleen cells of immunized rats10 and mice11.

  • The ELISPOT assay can be helpful in better understanding how pathogens evade protective immunity12

  • The ELISPOT assay is being used in the course of vaccination trials aimed at the induction of tumor-specific T cells13,14.


Flow diagram T cell ELISPOT assay

Brief description of the T cell ELISPOT procedure

Cells are incubated for a defined period of time in the wells of the ELISPOT plate coated with a high-affinity monoclonal antibody to which the cytokine, produced during incubation, will bind. Subsequently, cells are washed away. Areas in which the cytokine has been captured by the coating antibody are detected with a combination of biotinylated anti-cytokine detection antibodies and enzyme-labeled streptavidin. 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 T cell ELISPOT Flow diagram.

Example of the Human T cell ELISPOT assay:

COVID-19 vaccination T cell ELISPOT results

This is an example of IFN-γ and IL-2 specific spots produced by T cells in human PBMC.
The subject was vaccinated (booster vaccination) intramuscularly with a COVID-19 mRNA vaccine. PBMC from before and after vaccination were stimulated with a SARS-CoV-2 peptide pool and incubated for 24 hours on the ELISPOT plate (2x105 cells/well).

U-CyTech ELISPOT products

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ELISPOT applied in researchFAQs ELISPOT assayT cell ELISPOT guidelinesManual databaseReference databaseT cell ELISPOT productsOrdering information



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. 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.
  5. 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. Oct;36(5): 859-868
  6. 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.
  7. 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.
  8. 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.
  9. Karmakar S et al. (2014). Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons. Vaccine 32: 1296-303.
  10. Liu GX et al. (2009). Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats. Vaccine 27: 2940-7. 
  11. Wong YC et al. (2021). Sustained viremia suppression by SHIVSF162P3CN-recalled effector-memory CD8+ T cells after PD1-based vaccination. PLoS Pathog. 17(6): e1009647. 
  12. Li Z et al. (2022). Impaired T lymphocyte responses during childhood Staphylococcus aureus infection. J. Infect. Dis. 225: 177-85.
  13. 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.
  14. Li J et al. (2007) Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy. PloS One. 2:e1122.