Abstract
Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, “blank,” ELISPOT plate. After finishing the incubations, the amount of IFN gamma was measured by ELISA in culture media collected from both plates. The “blank” plate served to measure a total amount of secreted IFN gamma, whereas the ELISPOT plate served to measure the amount of unbound (UB) IFN gamma. Subtracting the amount of unbound IFN gamma from its total amount and dividing it by the number of spots in the ELISPOT plate allows for the calculation of the average amount of IFN gamma in a spot formed by a single cell.
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References
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.
Kalyuzhny, A. E. (2005) Chemistry and biology of the ELISPOT assay. Methods Mol Biol 302, 15–31.
Kalyuzhny, A. E. (2009) ELISPOT assay on membrane microplates. Methods Mol Biol 536, 355–365.
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.
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.
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-gamma (IFN-gamma)-secreting lymphocytes. Clin Exp Immunol 120, 476–482.
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.
Keane, N. M., Price, P., Stone, S. F., 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 antiretroviral therapy. AIDS Res Hum Retroviruses 16, 1991–1996.
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, 2285–2293.
Eriksson, K., Nordstrom, I., Horal, P., Jeansson, S., Svennerholm, B., Vahlne, A., et al. (1992) Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates. J Immunol Methods 153, 107–113.
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, 442–449.
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.
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.
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.
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, 93–97.
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Hagen, J., Houchins, J.P., Kalyuzhny, A.E. (2012). Combining ELISPOT and ELISA to Measure Amounts of Cytokines Secreted by a Single Cell. 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_10
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DOI: https://doi.org/10.1007/978-1-61779-325-7_10
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