The Ex Vivo IFN-γ Enzyme-Linked Immunospot (ELISpot) Assay

  • Martha SedegahEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1325)


The quantification of single cell interferon-gamma (IFN-γ) release for assessing cellular immune responses using the Enzyme-linked immunospot (ELISPOT) assay is an invaluable technique in immunology. Peripheral blood mononuclear cells (PBMC) are stimulated in vitro with recombinant proteins, peptides and recently whole malaria organisms. Stimulation may be short term (20–36 h) or long term (cultured ELISpot, up to 7 days). ELISpot is also able to quantify other cytokines secreted by antigen-specific T-cells, such as interleukin-2, interleukin-5, and other interleukins. ELISpot is playing an important role especially in vaccine research studies.

Key words

ELISPOT Peripheral blood mononuclear cells PBMC Interferon-gamma IFN-γ Cytokines T cells 



The following have made major contributions to the development and use of this assay: Harini Ganeshan, Maria Belmonte, Jun Huang, Esteban Abot, and Arnel Belmonte, and helped write this chapter with Michael R. Hollingdale.


  1. 1.
    Czerkinsky CC et al (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65(1–2):109–121CrossRefPubMedGoogle Scholar
  2. 2.
    Sedgwick JD, Holt PG (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods 57(1–3):301–309CrossRefPubMedGoogle Scholar
  3. 3.
    Janetzki S et al (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291(1–2):175–183CrossRefPubMedGoogle Scholar
  4. 4.
    Janetzki S et al (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51–86PubMedGoogle Scholar
  5. 5.
    Cox JH et al (2002) Accomplishing cellular immune assays for evaluation of vaccine efficacy in developing countries. In: Rose NR, Hamilton RG, Detrick B (eds) Manual clinical laboratory immunology. ASM Press, Washington, DC, pp 301–315Google Scholar
  6. 6.
    Scheibenbogen 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(1–2):81–89CrossRefPubMedGoogle Scholar
  7. 7.
    Janetzki S, Britten CM (2012) The impact of harmonization on ELISPOT assay performance. Methods Mol Biol 792:25–36CrossRefPubMedGoogle Scholar
  8. 8.
    Slota M et al (2011) ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines 10(3):299–306PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Zhang W, Lehmann PV (2012) Objective, user-independent ELISPOT data analysis based on scientifically validated principles. Methods Mol Biol 792:155–171CrossRefPubMedGoogle Scholar
  10. 10.
    Lehmann PV, Zhang W (2012) Unique strengths of ELISPOT for T cell diagnostics. Methods Mol Biol 792:3–23CrossRefPubMedGoogle Scholar
  11. 11.
    Sedegah M et al (2011) Adenovirus 5-vectored P. falciparum vaccine expressing CSP and AMA1. Part A: safety and immunogenicity in seronegative adults. PLoS One 6(10):e24586PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Sedegah M et al (2011) Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein. Malar J 9:241CrossRefGoogle Scholar
  13. 13.
    Dodoo D et al (2011) Measuring naturally acquired immune responses to candidate malaria vaccine antigens in Ghanaian adults. Malar J 10:168PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Tamminga C et al (2011) Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component. PLoS One 6(10):e25868PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Chuang I et al (2013) DNA prime/adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity. PLoS One 8(2):1371CrossRefGoogle Scholar
  16. 16.
    Sedegah M et al (2013) Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP). Malar J 12:185PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Rosenberg ES et al (1999) Characterization of HIV-1-specific T-helper cells in acute and chronic infection. Immunol Lett 66(1–3):89–93CrossRefPubMedGoogle Scholar
  18. 18.
    Epstein JE et al (2011) Live attenuated malaria vaccine designed to protect through hepatic CD8+ T Cell immunity. Science 334:475–480CrossRefPubMedGoogle Scholar
  19. 19.
    Seder RA et al (2013) Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341(6152):1359–1365CrossRefPubMedGoogle Scholar
  20. 20.
    Nielsen M et al (2003) Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12(5):1007–1017PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Rammensee H et al (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50(3–4):213–219CrossRefPubMedGoogle Scholar
  22. 22.
    Dodoo D et al (2008) Cohort study of the association of antibody levels to AMA1, MSP119, MSP3 and GLURP with protection from clinical malaria in Ghanaian children. Malar J 7:142PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Naval Medical Research CenterSilver SpringUSA

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