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Handbook of ELISPOT pp 51–86Cite as

Standardization and Validation Issues of the ELISPOT Assay

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 302))

Abstract

During the last 20 yr, the enzyme-linked immunospot (ELISPOT) assay has emerged as one of the most important and widely used assays to monitor immune responses in humans and a variety of other species. With the ELISPOT assay, immune cell frequencies can be measured at the single cell level without elaborate expansion or manipulation of cell populations. Its usefulness has led to its application in vaccine design and development and, most importantly, in monitoring vaccination efforts. The impact of results measured with this assay can be profound. In addition to ease of performance, repeatability and reliability are major features expected of an ELISPOT assay. The focus today is on standardization of the technique, validation strategies to comply with these required features, and accommodation of the growing demand of Good Laboratory Practice (GLP) compliance. This chapter will give the experienced scientists as well as newcomers to the field an overview over the major standardization issues for each step of the protocol. Guidelines are given on how to validate the ELISPOT performance.

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References

  1. Czerkinsky C. C, Nilsson L. A., Nygren H., Ouchterlony O., and Tarkowski A. (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109–121.

    Article  PubMed  CAS  Google Scholar 

  2. Sedgwick J. D., and Holt P. G. (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57, 301–309.

    Article  PubMed  CAS  Google Scholar 

  3. Czerkinsky C, Andersson G., Ekre H. P., Nilsson L. A., Klareskog L., and Ouchterlony 0. (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 

  4. Janetzki S., Palla D., Rosenhauer V., Lochs H., Lewis J. J., and Srivastava P. K. (2000) Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: a pilot study. Int. J. Cancer 88, 232–238.

    Article  PubMed  CAS  Google Scholar 

  5. Lewis J. J., Janetzki S., Schaed S., Panageas K. S., Wang S., Williams L., et al. (2000) Evaluation of CD8(+) T-cell frequencies by the ELISPOT assay in healthy individuals and in patients with metastatic melanoma immunized with tyrosinase peptide. Int. J. Cancer 87, 391–398.

    Article  PubMed  CAS  Google Scholar 

  6. Pass H. A., Schwarz S. L., Wunderlich J. R., and Rosenberg S. A. (1998) Immunization of patients with melanoma peptide vaccines: immunologic assessment using the ELISPOT assay. Cancer J. Sci. Am. 4, 316–323.

    PubMed  CAS  Google Scholar 

  7. Wang E, Bade E., Kuniyoshi C, Spears L., Jeffery G., Marty V., et al. (1999) Phase I trial of a MART-1 peptide vaccine with incomplete Freund’s adjuvant for resected high-risk melanoma. Clin. Cancer Res. 5, 2756–2765.

    PubMed  CAS  Google Scholar 

  8. Cox J., deSouza M., Ratto-Kim S., Ferrari G., Weinhold K., and Birx D. (2002) Accomplishing cellular immune assays for evaluation of vaccine efficacy in developing countries., in Manual of Clinical Laboratory Immunology ASM Press, Washingon, DC, pp. 301–315.

    Google Scholar 

  9. Lathey J. (2003) Preliminary steps toward validating a clinical bioassay. BioPharm. Int., 42–50.

    Google Scholar 

  10. Mwau M., McMichael A., and Hanke T. (2002) Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res. Hum. Retroviruses 18, 611–618.

    Article  PubMed  CAS  Google Scholar 

  11. 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 

  12. Cox J., Ferrari G., Kalams S. A., Lopaczynski W, Oden N., D’Souza P., and Group, a. t. E. C. S. (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international immunodeficiency virus type 1 vaccine trials. AIDS Res. & Hu. Retroviruses 21, in press.

    Google Scholar 

  13. Shaw R. D., Merchant A. A., Groene W. S., and Cheng E. H. (1993) Persistence of intestinal antibody response to heterologous rotavirus infection in a murine model beyond 1 year. J. Clin. Microbiol. 31, 188–191.

    PubMed  CAS  Google Scholar 

  14. Boyum A. (1968) isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, 77–89.

    Article  CAS  Google Scholar 

  15. 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 

  16. Russell N. D., Hudgens M. G., Ha R., Havenar-Daughton C, and McElrath M. J. (2003) Moving to HIV-1 vaccine efficacy trials: defining T-cell responses as potential correlates of immunity. J. Infect. Dis. 187, 226–242.

    Article  PubMed  CAS  Google Scholar 

  17. Smith J. G., Liu X., Kaufhold R. M., Clair J., and Caulfield M. J. (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, 871–879.

    PubMed  CAS  Google Scholar 

  18. Weinberg A. (2002) Cryopreservation of peripheral blood mononuclear cells, in Manual of Clinical Laboratory Immunology. ASM Press, Washington, DC, pp. 220–223.

    Google Scholar 

  19. Betensky R., Connick E., Devers J., Landay A., Nokta M., Plaeger S., et al. (2000) Shipment impairs lymphocyte proliferative responses to microbial antigens. Clin. Diagn. Lab. Immunol. 7, 759–763.

    PubMed  CAS  Google Scholar 

  20. Weinberg A., Betensky R., Zhang L., and Ray G. (1998) Effect of shipment, stor age, anticoagulant, and cell separation on lymphocyte proliferation assays for human immunodeficiency virus-infected patients. Clin. Diagn. Lab. Immunol. 5, 804–807.

    PubMed  CAS  Google Scholar 

  21. Currier J., Kuta E., Turk E., Earhart L., 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 

  22. Dudley M. E., Wunderlich J. R., Robbins P. E, Yang J. C, Hwu P., Schwartzentruber D. J., et al. (2002) Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298, 850–854.

    Article  PubMed  CAS  Google Scholar 

  23. Shankaran V., Ikeda H., Bruce A. T., White J. M., Swanson P. E., Old L. J., et al. (2001) IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 410, 1107–1111.

    Article  PubMed  CAS  Google Scholar 

  24. Yee C, Savage P. A., Lee P. P., Davis M. M., and Greenberg P. D. (1999) Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J. Immunol. 162, 2227–2234.

    PubMed  CAS  Google Scholar 

  25. Yee C, Thompson J. A., Byrd D., Riddell S. R., Roche P., Celis E., et al. (2002) Adoptive T-cell therapy using antigen-specific CD8+ T-cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T-cells. Proc. Natl. Acad. Sci. USA 99, 16168–16173.

    Article  PubMed  CAS  Google Scholar 

  26. Camara N. 0., Sebille R, and Lechler R. I. (2003) Human CD4+CD25+ regulatory cells have marked and sustained effects on CD8+ T-cell activation. Eur. J. Immunol. 33, 3473–3483.

    Article  PubMed  CAS  Google Scholar 

  27. Grakoui A., Shoukry N. H., Woollard D. J., Han J. H., Hanson H. L., Ghrayeb J., et al. (2003) HCV persistence and immune evasion in the absence of memory T-cell help. Science 302, 659–662.

    Article  PubMed  CAS  Google Scholar 

  28. Matloubian M., Concepcion R. J., and Ahmed R. (1994) CD4+ T-cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J. Virol. 68, 8056–8063.

    PubMed  CAS  Google Scholar 

  29. von Herrath M. G., Yokoyama M., Dockter J., Oldstone M. B., and Whitton J. L. (1996) CD4-deficient mice have reduced levels of memory cytotoxic T-lymphocytes after immunization and show diminished resistance to subsequent virus challenge. J. Virol. 70, 1072–1079.

    Google Scholar 

  30. Wei W. Z., Morris G. P., and Kong Y. C. (2004) Anti-tumor immunity and autoimmunity: a balancing act of regulatory T-cells. Cancer Immunol. Immunother. 53, 73–78.

    Article  PubMed  CAS  Google Scholar 

  31. Wherry E. J., Blattman J. N., Murali-Krishna K., van der Most R., and Ahmed R. (2003) Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77, 4911–4927.

    Article  PubMed  CAS  Google Scholar 

  32. Rosenberg E. S., LaRosa L., Flynn T., Robbins G., and Walker B. D. (1999) Characterization of HIV-1-specific T-helper cells in acute and chronic infection. Immunol. Lett. 66, 89–93.

    Article  PubMed  CAS  Google Scholar 

  33. Currier J. R., deSouza M., Chanbancherd P., Bernstein W, Birx D. L., and Cox J. H. (2002) Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD 8 cytotoxic T-lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and-C(W)0304 alleles. J. Virol. 76, 4971–4986.

    Article  PubMed  CAS  Google Scholar 

  34. Larsson M., Jin X., Ramratnam B., Ogg G. S., Engelmayer J., Demoitie M. A., et al. (1999) A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T-cells in HIV-1-positive individuals. Aids 13, 767–777.

    Article  PubMed  CAS  Google Scholar 

  35. McAdam S., Kaleebu P., Krausa P., Goulder P., French N., Collin B., et al. (1998) Cross-clade recognition of p55 by cytotoxic T-lymphocytes in HIV-1 infection. Aids 12, 571–579.

    Article  PubMed  CAS  Google Scholar 

  36. Pathan A. A., Wilkinson K. A., Wilkinson R. J., Latif M., McShane H., Pasvol G., et al. (2000) High frequencies of circulating IFN-gamma-secreting CD8 cytotoxic T-cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease. Eur. J. Immunol. 30, 2713–2721.

    Article  PubMed  CAS  Google Scholar 

  37. Wilson C. C, Palmer B., Southwood S., Sidney J., Higashimoto Y., Appella E., et al. (2001) Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes. J. Virol. 75, 4195–4207.

    Article  PubMed  CAS  Google Scholar 

  38. Draenert R., Altfeld M., Brander C, Basgoz N., Corcoran C, Wurcel A. G., et al. (2003) Comparison of overlapping peptide sets for detection of antiviral CD8 and CD4 T-cell responses. J. Immunol. Methods 275, 19–29.

    Article  PubMed  CAS  Google Scholar 

  39. Edwards B. H., Bansal A., Sabbaj S., Bakari J., Mulligan M. J., and Goepfert P. A. (2002) Magnitude of functional CD8+ T-cell responses to the gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma. J. Virol. 76, 2298–2305.

    Article  PubMed  CAS  Google Scholar 

  40. Ferrari G., Neal W., Jones A., Olender N., Ottinger J., Ha R., et al. (2001) CD8 CTL responses in vaccinees: emerging patterns of HLA restriction and epitope recognition. Immunol. Lett. 79, 37–45.

    Article  PubMed  CAS  Google Scholar 

  41. Kern E, Surel I. P., Faulhaber N., Frommel C, Schneider-Mergener J., Schonemann C, et al. (1999) Target structures of the CD8(+)-T-cell response to human cytomegalovirus: the 72-kilodalton major immediate-early protein revisited. J. Virol. 73, 8179–8184.

    PubMed  CAS  Google Scholar 

  42. Masemola A., Mashishi T., Khoury G., Mohube P., Mokgotho P., Vardas E., et al. (2004) Hierarchical targeting of subtype C HIV-1 proteins by CD8+ T-cells:correlation with viral load. J. Virol. 78, 3233–3243.

    Article  PubMed  CAS  Google Scholar 

  43. Novitsky V., Rybak N., McLane M. E, Gilbert P., Chigwedere P., Klein I., et al. (2001) Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific ELISPOT-based cytotoxic T-lymphocyte responses for AIDS vaccine design. J. Virol. 75, 9210–9228.

    Article  PubMed  CAS  Google Scholar 

  44. Ioannides C. (2003) Improving the accuracy and speed of mammalian cell counting. Am. Biotechnology Lab. May,, 10–12

    Google Scholar 

  45. Lem L. (2003) Cell counting and viability assessments in the process. Development of cellular therapeutics. BioProcessing J. July/August, 57–60.

    Google Scholar 

  46. Feldkamp C. S., and Carey J. L. (2002) Standardization of Immunoassay methodologies, in Manual of Clinical Laboratory Imunology (Rose N. R., Hamilton R.G., Detrick B., eds), ASM Press, Washington, DC pp. 1215–1226.

    Google Scholar 

  47. 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 

  48. Cui Y., and Chang L. J. (1997) Computer-assisted, quantitative cytokine enzymelinked immunospot analysis of human immune effector cell function. Biotechniques 22, 1146–1149.

    PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Vaquerano J. E., Peng M., Chang J. W., Zhou Y. M., and Leong S. P. (1998) Digital quantification of the enzyme-linked immunospot (ELISPOT). Biotechniques 25, 830–836.

    PubMed  CAS  Google Scholar 

  51. 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 

  52. Janetzki S., Schaed S., Blachere N. E. B., 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 

  53. Lewis C. E., McCracken D., Ling R., Richards P. S., McCarthy S. P., and McGee J. O. (1991) Cytokine release by single, immunophenotyped human cells: use of the reverse hemolytic plaque assay. Immunol. Rev. 119, 23–39.

    Article  PubMed  CAS  Google Scholar 

  54. Hesse M. D., Karulin A. Y, Boehm B. O., Lehmann P. V., and Tary-Lehmann M. (2001) A T-cell clone’s avidity is a function of its activation state. J. Immunol. 167, 1353–1361.

    PubMed  CAS  Google Scholar 

  55. Karulin A. Y, Hesse M. D., Tary-Lehmann M., and Lehmann P. V. (2000) Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity. J. Immunol. 164, 1862–1872.

    PubMed  CAS  Google Scholar 

  56. Bennouna J., Hildesheim A., Chikamatsu K., Gooding W., Storkus W. J., and Whiteside T. L. (2002) Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses. J. Immunol. Methods 261, 145-56.

    Google Scholar 

  57. Dunnet C. W. (1964) New table for multiple comparison with a control. Biometrics 20, 482–491.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  59. Hochberg Y, and Tamhane A. C. (1987) Multiple Comparison Procedures. Wiley, New York.

    Book  Google Scholar 

  60. Westfall P. H., and Young S. S. (1993) Resampling-Based Multiple Testing: Examples and Methods for P-Value Adjustment. Wiley, New York

    Google Scholar 

  61. Ewens W. J., and Grant G.R. (2001) Statistical Methods in Bioinformatics. Springer Verlag, New York, pp. 356–360.

    Google Scholar 

  62. Westfall P. H., and Tobias R.D. (1999) Multiple Comparisons and Multiple Tests Using the SAS System. SAS Publishing, Cary, NC.

    Google Scholar 

  63. Fleiss J. L. (1973) Statistical Methods for Rates and Proportions. Wiley, New York.

    Google Scholar 

  64. Landis J. R., and Koch G. G. (1977) The measurement of observer agreement for categorical data. Biometrics 33, 159–174.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. ZellNet Consulting Inc., Fort Lee, NJ

    Sylvia Janetzki

  2. The US Military HIV Research Program, Henry M. Jackson Foundation, Rockville, MD

    Josephine H. Cox

  3. The EMMES Corporation, Rockeville, MD

    Neal Oden

  4. Department of Surgery, Duke University, Durham, NC

    Guido Ferrari

Authors
  1. Sylvia Janetzki
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  2. Josephine H. Cox
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  3. Neal Oden
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  4. Guido Ferrari
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Editor information

Editors and Affiliations

  1. R&D Systems Inc.,, Minneapolis, MN

    Alexander E. Kalyuzhny

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© 2005 Humana Press Inc., Totowa, NJ

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Janetzki, S., Cox, J.H., Oden, N., Ferrari, G. (2005). Standardization and Validation Issues of the ELISPOT Assay. In: Kalyuzhny, A.E. (eds) Handbook of ELISPOT. Methods in Molecular Biology™, vol 302. Humana Press. https://doi.org/10.1385/1-59259-903-6:051

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  • DOI: https://doi.org/10.1385/1-59259-903-6:051

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-469-2

  • Online ISBN: 978-1-59259-903-5

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