Chemistry and Biology of the ELISPOT Assay

  • Alexander E. Kalyuzhny
Part of the Methods in Molecular Biology™ book series (MIMB, volume 302)

Abstract

Enzyme-linked immunospot, or ELISPOT, assay allows the detection of low frequencies of cells secreting various molecules. ELISPOT can be used in many areas of research and, because of its high sensitivity, has the potential to become a valuable diagnostic tool. Based on the same “sandwich” immunochemical principles as enzyme-linked immunosorbent assay, ELISPOT is easy to perform and quantify the results. At the same time ELISPOT remains a state-of-the-art technique that requires accuracy, thorough selection of antibodies and detection reagents, and an understanding of the principles of data analysis. This review covers various technical aspects of the ELISPOT assay, including immunochemical principles of the assay, selection of reagents and plates, and troubleshooting recommendations.

Key Words

ELISPOT detection antibodies capture antibodies spot-forming cells quantification of spots spot artifacts 

References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Czerkinsky C. C., Nilsson L. A., Nygren H., Ouchterlony O., and Tarkowski A. (2983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109–121.CrossRefGoogle Scholar
  3. 3.
    Czerkinsky C., Moldoveanu Z., Mestecky J., Nilsson L. A., and Ouchterlony O. (1988) A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells. J. Immunol. Methods 115, 31–37.PubMedCrossRefGoogle Scholar
  4. 4.
    Tanguay S. and Killion J. J. (1994) Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine Cytokine Res. 13, 259–263.PubMedGoogle Scholar
  5. 5.
    Bienvenu J., Monneret G., Fabien N., and Revillard J. P. (2000) The clinical usefulness of the measurement of cytokines. Clin. Chem. Lab. Med. 38, 267–285.PubMedCrossRefGoogle Scholar
  6. 6.
    Mashishi T. and Gray C. M. (2002) The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T-cell epitopes. Clin. Chem. Lab. Med. 40, 903–910.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedGoogle Scholar
  8. 8.
    Asai T., Storkus W. J., and Whiteside T. L. (200) Evaluation of the modified ELISPOT assay for γ interferon production in cancer patients receiving antitumor vaccines. Clin. Diagn. Lab. Immunol. 7, 145–154.PubMedGoogle Scholar
  9. 9.
    Kamath A. T., Groat N. L., Bean A. G., and Britton W. J. (2000) Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-γ (IFN-γ)-secreting lymphocytes. Clin. Exp. Immunol. 120, 476–482.PubMedCrossRefGoogle Scholar
  10. 10.
    Eriksson K., Nordstrom I., Horal P., Jeansson S., Svennerholm B., Vahlne A., Holmgren J., and Czerkinsky C. (1992) Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates. J. Immunol. Methods 153, 107–113.PubMedCrossRefGoogle Scholar
  11. 11.
    Howell D. M., Feldman S. B., Kloser P., and Fitzgerald-Bocarsly P. (1994) Decreased frequency of functional natural interferon-producing cells in peripheral blood of patients with the acquired immune deficiency syndrome. Clin. Immunol. Immunopathol. 71, 223–230.PubMedCrossRefGoogle Scholar
  12. 12.
    Schmittel A., Keilholz U., Thiel E., and Scheibenbogen C. (2000) Quantification of tumor-specific T lymphocytes with the ELISPOT assay. J. Immunother. 23, 289–295.PubMedCrossRefGoogle Scholar
  13. 13.
    Smith S. M., Brookes R., Klein M. R., Malin A. S., Lukey P. T., King A. S., et al. (2000) Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A. J. Immunol. 165, 7088–7095.PubMedGoogle Scholar
  14. 14.
    Pelfrey C. M., Cotleur A. C., Lee J. C., and Rudick R. A. (2002) Sex differences in cytokine responses to myelin peptides in multiple sclerosis. J. Neuroimmunol. 130,211–223.PubMedCrossRefGoogle Scholar
  15. 15.
    Schmid-Grendelmeier P., Altznauer F., Fischer B., Bizer C., Straumann A., Menz G., Blaser K., et al. (2002). Eosinophils express functional IL-13 in eosinophilic inflammatory diseases. J. Immunol. 169, 1021–1027.PubMedGoogle Scholar
  16. 16.
    Sho M., Sandner S. E., Najafian N., Salama A. D., Dong V., Yamada A., et al. (2002) Sayegh: new insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs. Ann. Surg. 236, 667–675.PubMedCrossRefGoogle Scholar
  17. 17.
    Kemeny D. M. (1997) Enzyme-linked immunoassays, in Immunochemistry 1 (Johnstone A. P., and Turner M. W., eds)Oxford University Press, Oxford. p. 147–175.Google Scholar
  18. 18.
    Savage M. D., Mattson G., Desai S., Nielander G. W., Morgensen S., and Conklin E. J. (1992) Avidin-Biotin Chemistry: A Handbook. Pierce Chemical Co., Rockford, IL, p. 467.Google Scholar
  19. 19.
    Okamoto Y., Abe T., Niwa T., Mizuhashi S., andNishida M. (1998) Development of a dual color enzyme-linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells. Immunopharmacology 39,107–116.PubMedCrossRefGoogle Scholar
  20. 20.
    Okamoto Y., Gotoh Y., Tokui H., Mizuno A., Kobayashi Y., and Nishida M. (2000) Characterization of the cytokine network at a single cell level in mice with collagen-induced arthritis using a dual color ELISPOT assay. J. Interferon. Cytokine Res. 20,55–61.PubMedCrossRefGoogle Scholar
  21. 21.
    Favre N., Bordmann G., and Rudin W. (1997) Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR. J. Immunol. Methods 204, 57–66.PubMedCrossRefGoogle Scholar
  22. 22.
    Herr W., Schneider J., Lohse A. W., Meyer zum Buschenfelde K. H., and Wolfel T. (1996) Detection and quantification of blood-derived CD8+ T-lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. J. Immunol. Methods 191, 131–142.PubMedCrossRefGoogle Scholar
  23. 23.
    Arlen P., Tsang K. Y., Marshall J. L., Chen A., Steinberg S. M., Poole D., et al. (2000) The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol. Immunother. 49, 517–529.PubMedCrossRefGoogle Scholar
  24. 24.
    Janetzki S., Song P., Gupta V., Lewis J. J., and Houghton A. N. (2000) Insect cells as HLA-restricted antigen-presenting cells for the IFN-γ elispot assay. J. Immunol. Methods 234, 1–12.PubMedCrossRefGoogle Scholar
  25. 25.
    Ronnelid J., and Klareskog L. (1997) A comparison between ELISPOT methods for the detection of cytokine producing cells: greater sensitivity and specificity using ELISA plates as compared to nitrocellulose membranes. J. Immunol. Methods 200, 17–26.PubMedCrossRefGoogle Scholar
  26. 26.
    Schielen P., van Rodijnen W., Tekstra J., Albers R., and Seinen W. (1995) Quantification of natural antibody producing B-cells in rats by an improved ELISPOT technique using the polyvinylidene diflouride membrane as the solid support. J. Immunol. Methods 188, 33–41.PubMedCrossRefGoogle Scholar
  27. 27.
    McCutcheon M., Wehner N., Wensky A., Kushner M., Doan S., Hsiao L., et al. (1997) A sensitive ELISPOT assay to detect low-frequency human T-lymphocytes. J. Immunol. Methods 210, 149–166.PubMedCrossRefGoogle Scholar
  28. 28.
    Taguchi T., McGhee J. R., Coffman R. L., Beagley K. W., Eldridge J. H., Takatsu K., et al. (1990) Detection of individual mouse splenic T-cells producing IFN-γ and IL-5 using the enzyme-linked immunospot (ELISPOT) assay. J. Immunol. Methods 128, 65–73.PubMedCrossRefGoogle Scholar
  29. 29.
    Klencke B., Matijevic M., Urban R. G., Lathey J. L., Hedley M. L., Berry M., et al. (2002) Encapsulated plasmid DNA treatment for human papillomavirus 16-asso-ciated anal dysplasia: a Phase I study of ZYC101. Clin. Cancer Res. 8. 1028–1037.PubMedGoogle Scholar
  30. 30.
    McGhee M. L., Ogawa T., Pitts A. M., Moldoveanu Z., Mestecky J., McGhee J. R., and Kiyono H. (1989) Cellular analysis of functional mononuclear cells from chronically inflamed gingival tissue. Reg. Immunol. 2, 103–110.PubMedGoogle Scholar
  31. 31.
    Merville P., Pouteil-Noble C., Wijdenes J., Potaux L., Touraine J. L., and Banchereau J. (1993) Detection of single cells secreting IFN-γ, IL-6, and IL-10 in irreversibly rejected human kidney allografts, and their modulation by IL-2 and IL-4. Transplantation 55, 639–646.PubMedCrossRefGoogle Scholar
  32. 32.
    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.PubMedCrossRefGoogle Scholar
  33. 33.
    Holst G. C. (1998) CCD Arrays, Cameras, and Displays. 2nd ed., Society of Photo-optical Instrumentation Engineers, Wintrer Park, FL.Google Scholar
  34. 34.
    Kalyuzhny A., and Stark S. (2001) A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISPOT assays. J. Immunol. Methods 257, p. 93–97.PubMedCrossRefGoogle Scholar
  35. 35.
    Merker R., Check I., and Hunter R. L. (1979) Use of cryopreserved cells in quality control of human lymphocyte assays: analysis of variation and limits of reproducibility in long-term replicate studies. Clin. Exp. Immunol. 38, p. 116–126.PubMedGoogle Scholar
  36. 36.
    Lewalle P., Rouas R., Lehmann F., and Martiat P. (2000) Freezing of dendritic cells, generated from cryopreserved leukaphereses, does not influence their ability to induce antigen-specific immune responses or functionally react to maturation stimuli. J. Immunol. Methods 240, 69–78.PubMedCrossRefGoogle Scholar
  37. 37.
    Keane N. M., Price P., Stone S. E, John M., Murray R. J., and French M. A. (2000) Assessment of immune function by lymphoproliferation underestimates lymphocyte functional capacity in HIV patients treated with highly active anti-retroviral therapy. AIDS Res. Hum. Retroviruses 16, p. 1991–1996.PubMedCrossRefGoogle Scholar
  38. 38.
    Smith J. G., Liu X., Kaufhold R. M., Clair J., and Caulfield M. J. (2001) Development and validation of a γ interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin. Diagn. Lab. Immunol. 8, p. 871–879.PubMedGoogle Scholar
  39. 39.
    Sobota V., Bubenik J., Indrova M., Vlk V., and Jakoubkova J. (1997) Use of cryopreserved lymphocytes for assessment of the immunological effects of interferon therapy in renal cell carcinoma patients. J. Immunol. Methods 203, p. 1–10.PubMedCrossRefGoogle Scholar
  40. 40.
    Bailey T., Stark S., Grant A., Hartnett C., Tsang M., and Kalyuzhny A. (2002) A multidonor ELISPOT study of IL-1beta, IL-2, IL-4, IL-6, IL-13, IFN-γ and TNF-alpha release by cryopreserved human peripheral blood mononuclear cells. J. Immunol. Methods 270, p. 171–182.PubMedGoogle Scholar
  41. 41.
    Venkataraman M. (1994) Effects of cryopreservation on immune responses: VII. Freezing induced enhancement of IL-6 production in human peripheral blood mononuclear cells. Cryobiology 31, 468–477.PubMedCrossRefGoogle Scholar
  42. 42.
    Venkataraman M. (1995) Effects of cryopreservation on immune responses. VIII. Enhanced secretion of interferon-γ by frozen human peripheral blood mononuclear cells. Cryobiology 32, 528–534.PubMedCrossRefGoogle Scholar
  43. 43.
    Chapman A. L., Munkanta M., Wilkinson K. A., Pathan A. A., Ewer K., Ayles H., et al. (2002) Rapid detection of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacterium tuberculosis-specific T-cells. Aids 16, p. 2285–2293.PubMedCrossRefGoogle Scholar
  44. 44.
    Jakobson E., Masjedi K., Ahlborg N., Lundeberg L., Karlberg A. T., and Scheynius A. (2002) Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis. Br. J. Dermatol. 147, p. 442–449.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Alexander E. Kalyuzhny
    • 1
  1. 1.R&D Systems Inc.Minneapolis

Personalised recommendations