Skip to main content
SpringerLink
Log in

Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

  • Protocol
  • First Online:
Book cover Handbook of ELISPOT

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

Abstract

ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.

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. 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  CAS  Google Scholar 

  2. Herr, W., Schneider, J., Lohse, A.W., Meyer zum Buschenfelde, K., and Wolfel, T. (1996) Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor a in response to HLA-A2.1-binding melanoma and viral peptide antigens. J Immunol Methods 191:131–142

    Google Scholar 

  3. Herr, W., Protzer, U., Lohse, A.W., Gerken, G., Meyer zum Buschenfelde, K.H., and Wolfel, T. (1998) 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

    Google Scholar 

  4. 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(11): e7972. doi:10.1371/journal.pone.0007972

    Article  PubMed  Google Scholar 

  5. Schloot, N.C., Meierhoff, G., Karlsson, F.M., Ott, P., Putnam, A.,and 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 

  6. Asai, T., Storkus, W.J. and Whiteside, T.L. (2000) Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol 7:145–154

    PubMed  CAS  Google Scholar 

  7. 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 

  8. Speiser, D.E., Pittet, M.J., Guillaume, P., Lubenow, N., Hoffman, E., Cerottini, J.C., et al. (2004) Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring. J Immunother 27:298–308

    Article  PubMed  CAS  Google Scholar 

  9. 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 20;244:81–89

    Google Scholar 

  10. 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 

  11. 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 

  12. 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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  14. 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(5):527–37

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  16. Janetzki, S., Schaed, S., Blachere, N.E., Ben-Porat, L., Houghton, A.N., Panageas, K.S. (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291(1–2):175–83

    Article  PubMed  CAS  Google Scholar 

  17. Ryan, J.E., Ovsyannikova, I.G., Dhiman, N., Pinsky, N.A., Vierkant, R.A., Jacobson, R.M., et al. (2005) Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells. Scand J Clin Lab Invest 65(8):681–9

    Article  PubMed  CAS  Google Scholar 

  18. 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(1):68–81

    Article  PubMed  CAS  Google Scholar 

  19. 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(10):1489–501

    Article  PubMed  CAS  Google Scholar 

  20. Dubey, S., Clair, J., Fu, T.M., Guan, L., Long, R., Mogg, R., et al. (2007) 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

    Article  PubMed  CAS  Google Scholar 

  21. Hudgens, M.G., Self, S.G., Chiu, Y.L., Russell, N.D., Horton, H. and McElrath, M.J. (2004) Statistical considerations for the design and analysis of the ELISpot assay in HIV-1 vaccine trials. J Immunol Methods 288:19–34

    Article  PubMed  CAS  Google Scholar 

  22. Moodie, Z., Huang, Y., Gu, L., Hural, J. and Self, S.G. (2006) Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials. J Immunol Methods 315:121–132

    Article  PubMed  CAS  Google Scholar 

  23. Herr, W., Linn, B., Leister, N., Wandel, E., Meyer zum Buschenfelde, K., and Wolfel, T. (1997) The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor spots in response to peptide antigens. J Immunol Methods 203:141–152

    Google Scholar 

  24. Westfall, P.H. and Young, S.S. (1993) Resampling-based multiple testing: examples and methods for p-value adjustment. John Wiley and Sons, New York

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Zoe Moodie

  2. Division of Biostatistics, Department of Environmental Medicine, New York University School of Medicine, New York, NY, USA

    Leah Price

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

    Sylvia Janetzki

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

    Cedrik M. Britten

Authors
  1. Zoe Moodie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leah Price
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sylvia Janetzki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cedrik M. Britten
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zoe Moodie .

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

Moodie, Z., Price, L., Janetzki, S., Britten, C.M. (2012). Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories. 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_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-325-7_15

  • 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