Advertisement

Mastering the Computational Challenges of Elispot Plate Evaluation

  • Sylvia JanetzkiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1808)

Abstract

Much has been written about Elispot and how to optimally run the assay for a wide variety of applications. But only a limited number of articles exist addressing the analysis step, the plate evaluation. Comparing that fact with the vast amount of analysis advise available for other single cell immune assay, for example, intracellular cytokine staining, the overall impression may be that Elispot evaluation is just simple enough to not require extensive elaboration and guidance. At first thought this appears reasonable because how difficult can it be counting colored spots on a white background. In addition, automated Elispot readers were already introduced more than 20 years ago (Herr et al., J Immunol Methods 203, 141–152, 1997), easing the strenuous load of manual counting and providing means to decrease the subjectivity in Elispot analysis. Just shortly thereafter however, the first report was published about the subjectivity and operator-dependency of plate evaluation even when using automated reader systems (Janetzki et al., J Immunol Methods 291, 175–183, 2004). Later, the plate evaluation was identified as a main factor causing variability in Elispot results, triggering the inclusion of recommendations on handling of artifacts and the audits of plate reading results in the Initial Elispot Harmonization guidelines (Janetzki et al., Cancer Immunol Immunother 57, 303–315, 2008; Britten et al., Cancer Immunol Immunother 57, 289–302, 2008). In follow-up, a large international study with 75 laboratories was conducted to address the current approaches taken to evaluate Elispot plates and to establish consensus guidelines for plate evaluation (Janetzki et al., Nat Protoc 10, 1098–1115, 2015). This article addresses the special challenges of plate evaluation, gives explanations for unusual observation, and provides overall recommendations on how to work through the labyrinth of available algorithms and reader settings to obtain reliable Elispot data.

Key words

Elispot Elispot evaluation Elispot reader Elispot images Elispot software Elispot analysis 

References

  1. 1.
    Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65:109–121CrossRefPubMedGoogle Scholar
  2. 2.
    Weiss AJ (2005) Membranes and membrane plates used in ELISPOT. Methods Mol Biol 302:33–50PubMedGoogle Scholar
  3. 3.
    Weiss AJ (2012) Overview of membranes and membrane plates used in research and diagnostic ELISPOT assays. Methods Mol Biol 792:243–256CrossRefPubMedGoogle Scholar
  4. 4.
    Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303–315CrossRefPubMedGoogle Scholar
  5. 5.
    Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J, Haas I, Mackensen A, Kollgaard T, thor Straten P, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanovic S, Wolfel T, van der Burg SH (2008) 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 57:289–302CrossRefPubMedGoogle Scholar
  6. 6.
    Mallone R, Mannering SI, Brooks-Worrell BM, Durinovic-Bello I, Cilio CM, Wong FS, Schloot NC (2011) 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 163:33–49CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Janetzki S, Britten CM (2012) The impact of harmonization on ELISPOT assay performance. Methods Mol Biol 792:25–36CrossRefPubMedGoogle Scholar
  8. 8.
    Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, D’Souza M (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retrovir 21:68–81CrossRefPubMedGoogle Scholar
  9. 9.
    Rountree W, Berrong M, Sanchez AM, Denny TN, Ferrari G (2016) Variability of the IFN-gamma ELISpot assay in the context of proficiency testing and bridging studies. J Immunol Methods 433:69–76CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Janetzki S, Price L, Britten CM, van der Burg SH, Caterini J, Currier JR, Ferrari G, Gouttefangeas C, Hayes P, Kaempgen E, Lennerz V, Nihlmark K, Souza V, Hoos A (2010) Performance of serum-supplemented and serum-free media in IFNgamma Elispot Assays for human T cells. Cancer Immunol Immunother 59:609–618CrossRefPubMedGoogle Scholar
  11. 11.
    van der Burg SH, Kalos M, Gouttefangeas C, Janetzki S, Ottensmeier C, Welters MJ, Romero P, Britten CM, Hoos A (2011) Harmonization of immune biomarker assays for clinical studies. Sci Transl Med 3:108ps144Google Scholar
  12. 12.
    ICH (1996.) I. C. o. H. Guidance for Industry: Q2B Validation of Analytical Procedures: MethodologyGoogle Scholar
  13. 13.
    Tuomela M, Stanescu I, Krohn K (2005) Validation overview of bio-analytical methods. Gene Ther 12(Suppl 1):S131–S138CrossRefPubMedGoogle Scholar
  14. 14.
    Janetzki S, Cox JH, Oden N, Ferrari G (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51–86PubMedGoogle Scholar
  15. 15.
    Mander A, Chowdhury F, Low L, Ottensmeier CH (2009) Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines. Cancer Immunol Immunother 58:789–800CrossRefPubMedGoogle Scholar
  16. 16.
    Smith JG, Liu X, Kaufhold RM, Clair J, Caulfield MJ (2001) Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin Diagn Lab Immunol 8:871–879PubMedPubMedCentralGoogle Scholar
  17. 17.
    Patton K, Aslam S, Lin J, Yu L, Lambert S, Dawes G, Esser MT, Woo J, Janetzki S, Cherukuri A (2014) Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells. Clin Vaccine Immunol 21:628–635CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sahin U, Derhovanessian E, Miller M, Kloke BP, Simon P, Lower M, Bukur V, Tadmor AD, Luxemburger U, Schrors B, Omokoko T, Vormehr M, Albrecht C, Paruzynski A, Kuhn AN, Buck J, Heesch S, Schreeb KH, Muller F, Ortseifer I, Vogler I, Godehardt E, Attig S, Rae R, Breitkreuz A, Tolliver C, Suchan M, Martic G, Hohberger A, Sorn P, Diekmann J, Ciesla J, Waksmann O, Bruck AK, Witt M, Zillgen M, Rothermel A, Kasemann B, Langer D, Bolte S, Diken M, Kreiter S, Nemecek R, Gebhardt C, Grabbe S, Holler C, Utikal J, Huber C, Loquai C, Tureci O (2017) Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547:222–226CrossRefPubMedGoogle Scholar
  19. 19.
    Bestard O, Crespo E, Stein M, Lucia M, Roelen DL, de Vaal YJ, Hernandez-Fuentes MP, Chatenoud L, Wood KJ, Claas FH, Cruzado JM, Grinyo JM, Volk HD, Reinke P (2013) Cross-validation of IFN-gamma Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients. Am J Transplant 13:1880–1890CrossRefPubMedGoogle Scholar
  20. 20.
    Tureci O, Vormehr M, Diken M, Kreiter S, Huber C, Sahin U (2016) Targeting the heterogeneity of cancer with individualized neoepitope vaccines. Clin Cancer Res 22:1885–1896CrossRefPubMedGoogle Scholar
  21. 21.
    Meier T, Eulenbruch HP, Wrighton-Smith P, Enders G, Regnath T (2005) 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 24:529–536CrossRefPubMedGoogle Scholar
  22. 22.
    Viklicky O, Hruba P, Tomiuk S, Schmitz S, Gerstmayer B, Sawitzki B, Miqueu P, Mrazova P, Tycova I, Svobodova E, Honsova E, Janssen U, Volk HD, Reinke P (2017) Sequential targeting of CD52 and TNF allows early minimization therapy in kidney transplantation: from a biomarker to targeting in a proof-of-concept trial. PLoS One 12:e0169624CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Janetzki S, Schaed S, Blachere NE, Ben-Porat L, Houghton AN, Panageas KS (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291:175–183CrossRefPubMedGoogle Scholar
  24. 24.
    Janetzki S, Price L, Schroeder H, Britten CM, Welters MJ, Hoos A (2015) Guidelines for the automated evaluation of Elispot assays. Nat Protoc 10:1098–1115CrossRefPubMedGoogle Scholar
  25. 25.
    Janetzki S (2016) Elispot for Rookies (And Experts Too). In: Kalyuzhny AE (ed) Techniques in life sciences and biomedicine for the non-expert. Springer, Cham, SwitzerlandGoogle Scholar
  26. 26.
    Schmielau J, Finn OJ (2001) Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res 61:4756–4760PubMedGoogle Scholar
  27. 27.
    De Rose R, Taylor EL, Law MG, van der Meide PH, Kent SJ (2005) Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate. J Immunol Methods 297:177–186CrossRefPubMedGoogle Scholar
  28. 28.
    McKenna KC, Beatty KM, Vicetti Miguel R, Bilonick RA (2009) Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function. J Immunol Methods 341:68–75CrossRefPubMedGoogle Scholar
  29. 29.
    Bull M, Lee D, Stucky J, Chiu YL, Rubin A, Horton H, McElrath MJ (2007) Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57–69CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Kierstead LS, Dubey S, Meyer B, Tobery TW, Mogg R, Fernandez VR, Long R, Guan L, Gaunt C, Collins K, Sykes KJ, Mehrotra DV, Chirmule N, Shiver JW, Casimiro DR (2007) Enhanced rates and magnitude of immune responses detected against an HIV vaccine: effect of using an optimized process for isolating PBMC. AIDS Res Hum Retrovir 23:86–92CrossRefPubMedGoogle Scholar
  31. 31.
    Janetzki S, Rabin R (2015) Enzyme-linked immunospot (elispot) for single-cell analysis. Methods Mol Biol 1346:27–46CrossRefPubMedGoogle Scholar
  32. 32.
    Janetzki S, Rueger M, Dillenbeck T (2014) Stepping up ELISpot: multi-level analysis in fluorospot assays. Cell 3:1102–1115CrossRefGoogle Scholar
  33. 33.
    Moodie Z, Price L, Gouttefangeas C, Mander A, Janetzki S, Lower M, Welters MJ, Ottensmeier C, van der Burg SH, Britten CM (2010) Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother 59:1489–1501CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Moodie Z, Price L, Janetzki S, Britten CM (2012) Response determination criteria for ELISPOT: toward a standard that can be applied across laboratories. Methods Mol Biol 792:185–196CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.ZellNet Consulting, Inc.Fort LeeUSA

Personalised recommendations