Protocol | Published:

Guidelines for the automated evaluation of Elispot assays

Nature Protocols volume 10, pages 10981115 (2015) | Download Citation


The presented protocol for Elispot plate evaluation summarizes how to implement the recommendations developed following the establishment of a large-scale international Elispot plate-reading panel and subsequent multistep consensus-finding process. The panel involved >100 scientists from various immunological backgrounds. The protocol includes the description and justification of steps for setting reading parameters to obtain accurate, reliable and precise automated analysis results of Elispot plates. Further, necessary adjustments for out-of-specification situations are described and examples are provided. The plate analysis, including parameter adjustments, auditing of results and necessary annotations, should be achievable within a time range of 10–30 min per plate. Adoption of these guidelines should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot. These guidelines conclude the ongoing harmonization efforts for the enzymatic Elispot assay.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , , , & A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109–121 (1983).

  2. 2.

    , & The role of IFN-γ Elispot assay in HIV vaccine research. Nat. Protoc. 4, 461–469 (2009).

  3. 3.

    , , , & Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood. Nat. Protoc. 8, 1073–1087 (2013).

  4. 4.

    , , , & Sensitivity of a new commercial enzyme-linked immunospot assay (T SPOT-TB) for diagnosis of tuberculosis in clinical practice. Eur. J. Clin. Microbiol. Infect Dis. 24, 529–536 (2005).

  5. 5.

    et al. Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat. Commun. 4, 2836 (2013).

  6. 6.

    et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N. Engl. J. Med. 361, 1838–1847 (2009).

  7. 7.

    et al. Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nat. Med. 18, 1254–1261 (2012).

  8. 8.

    et al. Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer. Cancer Immunol. Immunother. 62, 137–147 (2013).

  9. 9.

    , , & Triple cytokine FluoroSpot analysis of human antigen-specific IFN-γ, IL-17A and IL-22 responses. Cells 3, 1116–1130 (2014).

  10. 10.

    et al. A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines. J. Immunol. Methods 283, 91–98 (2003).

  11. 11.

    et al. Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol. Immunother. 57, 303–315 (2008).

  12. 12.

    et al. Harmonization of immune biomarker assays for clinical studies. Sci. Transl. Med. 3, 108ps144 (2011).

  13. 13.

    et al. The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol. Immunother. 57, 289–302 (2008).

  14. 14.

    & The impact of harmonization on ELISPOT assay performance. Methods Mol. Biol. 792, 25–36 (2012).

  15. 15.

    & How ELISPOT morphology reflects on the productivity and kinetics of cells′ secretory activity. Methods Mol. Biol. 792, 125–143 (2012).

  16. 16.

    et al. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J. Immunol. Methods 110, 29–36 (1988).

  17. 17.

    , , & Standardization and validation issues of the ELISPOT assay. Methods Mol. Biol. 302, 51–86 (2005).

  18. 18.

    et al. Improvement of IFN-γ ELISPOT performance following overnight resting of frozen PBMC samples confirmed through rigorous statistical analysis. Cells 4, 1–18 (2014).

  19. 19.

    et al. Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res. Hum. Retroviruses 21, 68–81 (2005).

  20. 20.

    et al. Evaluation of Elispot assays: influence of method and operator on variability of results. J. Immunol. Methods 291, 175–183 (2004).

  21. 21.

    et al. Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis. PLoS ONE 4, e7972 (2009).

  22. 22.

    et al. T cell assays and MIATA: the essential minimum for maximum impact. Immunity 37, 1–2 (2012).

  23. 23.

    et al. Response definition criteria for ELISPOT assays revisited. Cancer Immunol. Immunother. 59, 1489–1501 (2010).

  24. 24.

    , , & Single-cell technologies for monitoring immune systems. Nat. Immunol. 15, 128–135 (2014).

  25. 25.

    , , & Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol. 32, 684–692 (2014).

  26. 26.

    , & Intracellular cytokine optimization and standard operating procedure. Nat. Protoc. 1, 1507–1516 (2006).

  27. 27.

    , , , & Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1, 1522–1530 (2006).

  28. 28.

    et al. A harmonized approach to intracellular cytokine staining gating: results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI). Cytometry A 83, 728–738 (2013).

  29. 29.

    , & Stepping up ELISpot: multi-level analysis in FluoroSpot assays. Cells 3, 1102–1115 (2014).

  30. 30.

    et al. The effect of apoptotic cells on virus-specific immune responses detected using IFN-γ ELISPOT. J. Immunol. Methods 357, 51–54 (2010).

  31. 31.

    et al. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin. Exp. Immunol. 163, 33–49 (2011).

  32. 32.

    et al. Overnight resting of PBMC changes functional signatures of antigen specific T cell responses: impact for immune monitoring within clinical trials. PLoS ONE 8, e76215 (2013).

  33. 33.

    et al. Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8+ T cells with detection by ELISPOT and HLA-multimer staining. Cancer Immunol. Immunother. 63, 1199–1211 (2014).

  34. 34.

    et al. Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses. J. Immunol. Methods 391, 50–59 (2013).

  35. 35.

    et al. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity. J. Transl. Med. 1, 14 (2003).

  36. 36.

    & Enzyme-linked ImmunoSpot (ELISpot) for single cell analysis. Methods Mol. Biol. in the press (2015).

  37. 37.

    , , , & Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate. J. Immunol. Methods 297, 177–186 (2005).

  38. 38.

    et al. Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative persons at high risk for recent HIV exposure. J. Infect. Dis. 178, 260–265 (1998).

  39. 39.

    et al. Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay. J. Acquir. Immune. Defic. Syndr. 45, 20–27 (2007).

  40. 40.

    , , & Response determination criteria for ELISPOT: toward a standard that can be applied across laboratories. Methods Mol. Biol. 792, 185–196 (2012).

  41. 41.

    et al. Harmonization of the intracellular cytokine staining assay. Cancer Immunol. Immunother. 61, 967–978 (2012).

  42. 42.

    & Drug development: raise standards for preclinical cancer research. Nature 483, 531–533 (2012).

  43. 43.

    et al. A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J. Immunol. Methods 260, 157–172 (2002).

  44. 44.

    et al. Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J. Immunol. Methods 255, 27–40 (2001).

  45. 45.

    et al. Performance of serum-supplemented and serum-free media in IFN-γ Elispot assays for human T cells. Cancer Immunol. Immunother. 59, 609–618 (2010).

  46. 46.

    , & HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40, 693–696 (2000).

  47. 47.

    Overview of membranes and membrane plates used in research and diagnostic ELISPOT assays. Methods Mol. Biol. 792, 243–256 (2012).

Download references


C.M.B. and H.S. were supported by a grant from the Wallace Coulter Foundation (Florida, USA). We thank all participants in the Elispot plate reading panel and consensus process. The full list of participants, including several authors of this paper, is given in the Supplementary Note. We also thank A. Szterenfeld for helpful comments on the manuscript.

Author information

Author notes

    • Cedrik M Britten

    Present address: Immuno-Oncology and Combinations DPU, Oncology R&D, GlaxoSmithKline, Stevenage, UK.


  1. ZellNet Consulting, Inc., Fort Lee, New Jersey, USA.

    • Sylvia Janetzki
  2. LBPrice Statistical Consulting Ltd., Karmiel, Israel.

    • Leah Price
  3. BioNTech Diagnostics GmbH, Mainz, Germany.

    • Helene Schroeder
  4. Association for Cancer Immunotherapy (CIMT), Mainz, Germany.

    • Cedrik M Britten
  5. Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.

    • Marij J P Welters
  6. Department of ImmunoOncology, GlaxoSmith Kline, Collegeville, Pennsylvania, USA.

    • Axel Hoos


  1. Search for Sylvia Janetzki in:

  2. Search for Leah Price in:

  3. Search for Helene Schroeder in:

  4. Search for Cedrik M Britten in:

  5. Search for Marij J P Welters in:

  6. Search for Axel Hoos in:


S.J., L.P., C.M.B. and M.J.P.W. designed the study. S.J., L.P., H.S., C.M.B., M.J.P.W. and A.H. organized and led the panel. All panelists listed under acknowledgement provided data and feedback leading to the final protocol. S.J. and L.P. compiled and analyzed the data. S.J. wrote the manuscript.

Competing interests

S.J. is founder and president of ZellNet Consulting, Inc. L.P. is the founder and director of LBPrice Statistical Consulting, Ltd. All other authors declare no competing financial interests.

Corresponding author

Correspondence to Sylvia Janetzki.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Data, Supplementary Table 1 and Supplementary Note

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.