Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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):109–121
Sedgwick JD, Holt PG (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods 57(1–3):301–309
Janetzki S et al (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291(1–2):175–183
Janetzki S et al (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51–86
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 301–315
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):81–89
Janetzki S, Britten CM (2012) The impact of harmonization on ELISPOT assay performance. Methods Mol Biol 792:25–36
Slota M et al (2011) ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines 10(3):299–306
Zhang W, Lehmann PV (2012) Objective, user-independent ELISPOT data analysis based on scientifically validated principles. Methods Mol Biol 792:155–171
Lehmann PV, Zhang W (2012) Unique strengths of ELISPOT for T cell diagnostics. Methods Mol Biol 792:3–23
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):e24586
Sedegah M et al (2011) Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein. Malar J 9:241
Dodoo D et al (2011) Measuring naturally acquired immune responses to candidate malaria vaccine antigens in Ghanaian adults. Malar J 10:168
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):e25868
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):1371
Sedegah M et al (2013) Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP). Malar J 12:185
Rosenberg ES et al (1999) Characterization of HIV-1-specific T-helper cells in acute and chronic infection. Immunol Lett 66(1–3):89–93
Epstein JE et al (2011) Live attenuated malaria vaccine designed to protect through hepatic CD8+ T Cell immunity. Science 334:475–480
Seder RA et al (2013) Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341(6152):1359–1365
Nielsen M et al (2003) Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12(5):1007–1017
Rammensee H et al (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50(3–4):213–219
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 7:142
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sedegah, M. (2015). The Ex Vivo IFN-γ Enzyme-Linked Immunospot (ELISpot) Assay. In: Vaughan, A. (eds) Malaria Vaccines. Methods in Molecular Biology, vol 1325. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2815-6_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2815-6_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2814-9
Online ISBN: 978-1-4939-2815-6
eBook Packages: Springer Protocols