Skip to main content
SpringerLink
Log in

The Impact of Harmonization on ELISPOT Assay Performance

  • Protocol
  • First Online:
Handbook of ELISPOT

Part of the book series: Methods in Molecular Biology ((MIMB,volume 792))

Abstract

During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289–295, 2000; Whiteside Immunol Invest 29:149–162, 2000; Nagata et al. Ann N Y Acad Sci 1037:10–15, 2004; Cox et al. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC; Cox et al. Methods 38:274–282, 2006]. While the principles of the original protocol have changed little since its first introduction [Czerkinsky J Immunol Methods 110:29–36, 1988], individual laboratories have adapted assay procedures based on experimental needs, availability of reagents and equipment, obtained recommendations, and gained experience, leading to a wide disparity of applied ELISPOT protocols with inevitable consequences. This chapter addresses the resulting challenges for ELISPOT use in clinical trial settings, and discusses the influence of harmonization strategies as a tool for overcoming these challenges. Furthermore, harmonization is discussed in the context of assay standardization and validation strategies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kalyuzhny, A. E., (Ed.) (2005) Handbook of Elispot: Methods and Protocols, Vol. 302, Humana Press Inc., Totowa, NJ.

    Google Scholar 

  2. Schmittel, A., Keilholz, U., Thiel, E., and Scheibenbogen, C. (2000) Quantification of tumor-specific T lymphocytes with the ELISPOT assay., J Immunother 23, p289–295.

    Google Scholar 

  3. Whiteside, T. L. (2000) Immunologic monitoring of clinical trials in patients with cancer: technology versus common sense, Immunol Invest 29, 149–162.

    Article  PubMed  CAS  Google Scholar 

  4. Nagata, M., Kotani, R., Moriyama, H., Yokono, K., Roep, B. O., and Peakman, M. (2004) Detection of autoreactive T cells in type 1 diabetes using coded autoantigens and an immunoglobulin-free cytokine ELISPOT assay: report from the fourth immunology of diabetes society T cell workshop, Ann N Y Acad Sci 1037, 10–15.

    Article  PubMed  CAS  Google Scholar 

  5. Cox, J. H., D’Souza, M., Ratto-Kim, S., Ferrari, G., Weinhold, K., and Birx, D. L. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC.

    Google Scholar 

  6. Cox, J. H., Ferrari, G., and Janetzki, S. (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay, Methods 38, 274–282.

    Article  PubMed  CAS  Google Scholar 

  7. Czerkinsky, C., Andersson, G., Ekre, H. P., Nilsson, L. A., Klareskog, L., and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells, J Immunol Methods 110, 29–36.

    Article  PubMed  CAS  Google Scholar 

  8. Helms, T., Boehm, B. O., Asaad, R. J., Trezza, R. P., Lehmann, P. V., and Tary-Lehmann, M. (2000) Direct Visualization of Cytokine-Producing Recall Antigen-Specific CD4 Memory T Cells in Healthy Individuals and HIV Patients, J Immunol 164, 3723–3732.

    PubMed  CAS  Google Scholar 

  9. Janetzki, S., Cox, J. H., Oden, N., and Ferrari, G. (2005) Standardization and validation issues of the ELISPOT assay, Methods Mol Biol 302, 51–86.

    PubMed  CAS  Google Scholar 

  10. Maecker, H. T., Hassler, J., Payne, J. K., Summers, A., Comatas, K., Ghanayem, et al. (2008) Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides, BMC Immunol 9, 9.

    Google Scholar 

  11. Prabhakar, U., and Kelley, M. (2008) Validation of cell-based assays in the GLP setting: A practical guide. John Wiley, Chichester, UK.

    Google Scholar 

  12. Kreher, C. R., Dittrich, M. T., Guerkov, R., Boehm, B. O., and Tary-Lehmann, M. (2003) CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays, J Immunol Methods 278, 79–93.

    Article  PubMed  CAS  Google Scholar 

  13. Maecker, H. T., Dunn, H. S., Suni, M. A., Khatamzas, E., Pitcher, C. J., Bunde, T., et al. (2001) Use of overlapping peptide mixtures as antigens for cytokine flow cytometry, J Immunol Methods 255, 27–40.

    Article  PubMed  CAS  Google Scholar 

  14. Currier, J. R., Kuta, E. G., Turk, E., Earhart, L. B., Loomis-Price, L., Janetzki, S., et al. (2002) 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.

    Article  PubMed  CAS  Google Scholar 

  15. Smith, J. G., Joseph, H. R., Green, T., Field, J. A., Wooters, M., Kaufhold, R. M., et al. (2007) Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions, Clin Vaccine Immunol 14, 527–537.

    Article  PubMed  CAS  Google Scholar 

  16. Gazagne, A., Claret, E., Wijdenes, J., Yssel, H., Bousquet, F., Levy, E., et al. (2003) A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines, J Immunol Methods 283, 91–98.

    Article  PubMed  CAS  Google Scholar 

  17. Boulet, S., Ndongala, M. L., Peretz, Y., Boisvert, M. P., Boulassel, M. R., Tremblay, C., et al. (2007) A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses, J Immunol Methods 320, 18–29.

    Article  PubMed  CAS  Google Scholar 

  18. Shafer-Weaver, K., Rosenberg, S., Strobl, S., Gregory Alvord, W., Baseler, M., and Malyguine, A. (2006) Application of the granzyme B ELISPOT assay for monitoring cancer vaccine trials, J Immunother 29, 328–335.

    Article  PubMed  CAS  Google Scholar 

  19. Zuber, B., Levitsky, V., Jonsson, G., Paulie, S., Samarina, A., Grundstrom, et al. (2005) Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells, J Immunol Methods 302, 13–25.

    Google Scholar 

  20. Kalos, M. (2010) An integrative paradigm to impart quality to correlative science, J Transl Med 8, 26.

    Article  PubMed  Google Scholar 

  21. Janetzki, S., Schaed, S., Blachere, N. E., Ben-Porat, L., Houghton, A. N., and Panageas, K. S. (2004) Evaluation of Elispot assays: influence of method and operator on variability of results, J Immunol Methods 291, 175–183.

    Article  PubMed  CAS  Google Scholar 

  22. NCCLS. (2004) Performance of Single Cell Immune Response Assays; Approved Guideline., In NCCLS document I/LA26-A, NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA, USA.

    Google Scholar 

  23. Janetzki, S., Panageas, K. S., Ben-Porat, L., Boyer, J., Britten, C. M., Clay, T. M., et al. (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, 303–315.

    Article  PubMed  Google Scholar 

  24. Janetzki, S., Britten, C. M., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J. M., et al. (2009) “MIATA”-minimal information about T cell assays, Immunity 31, 527–528.

    Article  PubMed  CAS  Google Scholar 

  25. Cox, J. H., Ferrari, G., Kalams, S. A., Lopaczynski, W., Oden, N., and D’Souza M, P. (2005) 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.

    Google Scholar 

  26. Britten, C. M., Gouttefangeas, C., Welters, M. J., Pawelec, G., Koch, S., Ottensmeier, C., et al. (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, 289–302.

    Article  PubMed  CAS  Google Scholar 

  27. Boaz, M. J., Hayes, P., Tarragona, T., Seamons, L., Cooper, A., Birungi, J., et al. (2009) Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents, Clin Vaccine Immunol 16, 147–155.

    Article  PubMed  CAS  Google Scholar 

  28. Scheibenbogen, C., Romero, P., Rivoltini, L., Herr, W., Schmittel, A., Cerottini, J. C., et al. (2000) Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial, J Immunol Methods 244, 81–89.

    Article  PubMed  CAS  Google Scholar 

  29. Schloot, N. C., Meierhoff, G., Karlsson Faresjo, M., Ott, P., Putnam, A., Lehmann, P., 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, 365–376.

    CAS  Google Scholar 

  30. Smith, S. G., Joosten, S. A., Verscheure, V., Pathan, A. A., McShane, H., Ottenhoff, T. H., et al. (2009) Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis, PLoS One 4, e7972.

    Article  PubMed  Google Scholar 

  31. Britten, C. M., Janetzki, S., Ben-Porat, L., Clay, T. M., Kalos, M., Maecker, H., et al. (2009) Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium, Cancer Immunol Immunother 58, 1701–1713.

    Article  PubMed  CAS  Google Scholar 

  32. Britten, C. M., Janetzki, S., van der Burg, S. H., Gouttefangeas, C., and Hoos, A. (2008) Toward the harmonization of immune monitoring in clinical trials: quo vadis?, Cancer Immunol Immunother 57, 285–288.

    Article  PubMed  CAS  Google Scholar 

  33. Hoos, A., Eggermont, A., Janetzki, S., Hodi, S., Ibrahim, R., Andersen, A., et al. (2010) Improved Endpoints for Cancer Immunotherapy Trials, J Natl Cancer Inst 102,1388–1397.

    Google Scholar 

  34. Mander, A., Gouttefangeas, C., Ottensmeier, C., Welters, M. J., Low, L., van der Burg, S. H., et al. (2010) Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT Immunoguiding Program, Cancer Immunol Immunother 59, 619–627.

    Article  PubMed  CAS  Google Scholar 

  35. Janetzki, S., Price, L., Britten, C. M., van der Burg, S. H., Caterini, J., Currier, J. R., et al. (2010) Performance of serum-supplemented and serum-free media in IFNgamma Elispot Assays for human T cells, Cancer Immunol Immunother 59, 609–618.

    Article  PubMed  CAS  Google Scholar 

  36. Moodie, Z., Price, L., Gouttefangeas, C., Mander, A., Janetzki, S., Lower, M., et al. (2010) Response definition criteria for ELISPOT assays revisited, Cancer Immunol Immunother 59, 1489–1501.

    Article  PubMed  CAS  Google Scholar 

  37. The MIATA Project. http://www.miataproject.org. Accessed July 27, 2011.

Download references

Author information

Authors and Affiliations

  1. ZellNet Consulting, Inc., Fort Lee, NJ, USA

    Sylvia Janetzki

  2. Department of Internal Medicine, University Medical Center, Johannes Gutenberg-University Mainz III, Mainz, Germany

    Cedrik M. Britten

Authors
  1. Sylvia Janetzki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cedrik M. Britten
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sylvia Janetzki .

Editor information

Editors and Affiliations

  1. R & D Systems, Inc., McKinley Place NE. 614, Minneapolis, 55413, Minnesota, USA

    Alexander E. Kalyuzhny

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Janetzki, S., Britten, C.M. (2012). The Impact of Harmonization on ELISPOT Assay Performance. In: Kalyuzhny, A. (eds) Handbook of ELISPOT. Methods in Molecular Biology, vol 792. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-325-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-325-7_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-324-0

  • Online ISBN: 978-1-61779-325-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation