ELISPOT Assay to Detect Cytokine‐Secreting Murine and Human Cells

Dennis Klinman1

1 Cancer and Inflammation Program, National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.19
DOI:  10.1002/0471142735.im0619s83
Online Posting Date:  November, 2008
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Enzyme‐linked immunospot assays (ELISPOT) were initially developed to detect and quantify individual antibody‐secreting B cells. As the sensitivity of this assay improved, the much smaller amounts of cytokine and chemokine produced by individual T cells became amenable to detection. ELISPOT assays utilize high‐affinity antibody pairs directed against different epitopes on a single cytokine/chemokine. The critical first step involves binding the highest affinity Ab to a solid matrix. The plates are blocked to prevent nonspecific interactions, and the cells of interest incubated in the Ab‐coated wells, during which time they secrete the cytokine/chemokine of interest. The secreted protein binds to the Abs immediately below the producer cell. This bound protein is recognized by a secondary enzyme‐linked Ab. A colorimetric substrate is used to generate a dark precipitate or “spot” that marks the position of the protein‐producing cell. The resultant spots are permanent and can be quantified visually, microscopically, or electronically. Curr. Protoc. Immunol. 83:6.19.1‐6.19.9. © 2008 by John Wiley & Sons, Inc.

Keywords: assay; cytokine; chemokine; ELISPOT; single cell; T cell

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Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Cytokine ELISPOT Protocol
  • Alternate Protocol 1: Dual ELISPOT Assay for the Simultaneous Detection of Cells Secreting Two Different Cytokines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Basic Cytokine ELISPOT Protocol

  • Cytokine‐specific primary antibody diluted in carbonate buffer, pH 9.6
  • Blocking solution: PBS containing 2% to 5% (w/v) BSA, nonfat dry milk or FBS
  • Wash solution: PBS containing 0.25% (v/v) Tween 20
  • Distilled water
  • Incubation medium: RPMI‐1640 supplemented with 2.5% FBS (for the short duration of most incubations, other additives are not necessary, but can be included)
  • ELISPOT dilution buffer: PBS containing 1% (w/v) BSA
  • Labeled secondary cytokine‐specific antibody in ELISPOT dilution buffer
  • BCIP/NBT solution (Kirkegaard & Perry; prepare fresh according to manufacturer's instructions)
  • Aminoethylcarbazole (AEC) solution (see recipe)
  • 96‐well Immulon 2HB flat‐bottomed microtiter plates (Thermo Electron Corp).
  • Humidified 37°C, 5% CO 2 incubator
  • Dissecting microscope
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Literature Cited

   Altfeld, M.A., Trocha, A., Eldridge, R.L., Rosenberg, E.S., Phillips, M.N., Addo, M.M., Sekaly, R.P., Kalams, S.A., Burchett, S.A., McIntosh, K., Walker, B.D., and Goulder, P.J. 2000. Identification of dominant optimal HLA‐B60‐ and HLA‐B61‐restricted cytotoxic T‐lymphocyte CTL epitopes: Rapid characterization of CTL responses by enzyme‐linked immunospot assay. J. Virol. 74:8541‐8549.
   Boulet, S., Ndongala, M.L., Peretz, Y., Boisvert, M.P., Boulassel, M.R., Tremblay, C., Routy, J.P., Sekaly, R.P., and Bernard, N.F. 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.
   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.
   Czerkinsky, C., Andersson, G., Ekre, H.P., Nilsson, L.A., Klareskog, L., and Ouchterlony, O. 1988a. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma‐interferon‐secreting cells. J. Immunol. Methods 110:29‐36.
   Czerkinsky, C., Moldoveanu, Z., Mestecky, J., Nilsson, L.A., and Ouchterlony, O. 1988b. A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody‐secreting cells. J. Immunol. Methods 115:31‐37.
   Goulder, P.J., Brander, C., Annamalai, K., Mngqundaniso, N., Govender, U., Tang, Y., He, S., Hartman, K.E., O'Callaghan, C.A., Ogg, G.S., Altfeld, M.A., Rosenberg, E.S., Cao, H., Kalams, S.A., Hammond, M., Bunce, M., Pelton, S.I., Burchett, S.A., McIntosh, K., Coovadia, H.M., and Walker, B.D. 2000. Differential narrow focusing of immunodominant human immunodeficiency virus gag‐specific cytotoxic T‐lymphocyte responses in infected African and caucasoid adults and children. J. Virol. 74:5679‐5690.
   King, C.L., Poindexter, R.W., Ragunathan, J., Fleisher, T.A., Ottesen, E.A., and Nutman, T.B. 1991. Frequency analysis of IgE‐secreting B lymphocytes in persons with normal or elevated serum IgE levels. J. Immunol. 146:1478‐1483.
   King, C.L., Low, C.C., and Nutman, T.B. 1993. IgE production in human helminth infection. Reciprocal interrelationship between IL‐4 and IFN‐gamma. J. Immunol. 150:1873‐1880.
   Mahanty, S., Abrams, J.S., King, C.L., Limaye, A.P., and Nutman, T.B. 1992. Parallel regulation of IL‐4 and IL‐5 in human helminth infections. J. Immunol. 148:3567‐3571.
   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.
   Moller, S.A. and Borrebaeck, C.A. 1985. A filter immuno‐plaque assay for the detection of antibody‐secreting cells in vitro. J. Immunol. Methods 79:195‐204.
   Rönnelid, J. and Klareskog, L. 1997. A comparison between ELISPOT methods for the detection of cytokine producing cells. J. Immunol. Methods 200:17‐26.
   Rowland‐Jones, S.L., Dong, T., Fowke, K.R., Kimani, J., Krausa, P., Newell, H., Blanchard, T., Ariyoshi, K., Oyugi, J., Ngugi, E., Bwayo, J., MacDonald, K.S., McMichael, A.J., and Plummer, F.A. 1998. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV‐resistant prostitutes in Nairobi. J. Clin. Invest. 102:1758‐1765.
   Schmittel, A., Keilholz, U. and Scheibenbogen, C. 1997 Evaluation of the interferon‐gamma ELISPOT‐assay for quantification of peptide specific T lymphocytes from peripheral blood J. Immunol. Methods 210:167‐174.
   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.
   Shirai, A., Holmes, K., and Klinman, D.M. 1993. Detection and quantitation of cells secreting IL‐6 under physiologic conditions in BALB/c mice. J. Immunol. 150:793‐799.
   Shirai, A., Sierra, V., Kelly, C.I. and Klinman, D.M. 1994 Individual cells simultaneously produce both IL‐4 and IL‐6 under physiologic conditions in vivo. Cytokine 6:329‐336.
Key Reference
   Czerkinsky, C., Anderson, G., Ferrua, B., Nordstrom, I., Quiding, M., Eriksson, K., Larson, L., Hellstrand, K., and Ekre, H. 1991. Detection of human cytokine‐secreting cells in distinct anatomical compartments. Immunol. Rev. 119:5‐22.
  Provides useful overview of available cytokine ELISPOT assays.
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