Abstract
Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells.
The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and perforin Elispot assays are described.
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Acknowledgments
This work was supported by Progetto Strategico Regione Puglia grant (E.R., 2008), Ministero dell’Istruzione, dell’Università e della Ricerca (MiUR) FIRB, CAROMICS grant (E.R., 2011), and a Postdoctoral Fellowship by MiUR, PROGRAMMA OPERATIVO NAZIONALE (PON) RICERCA E COMPETITIVITÀ 2007–2013 PONa3_00395 “BIOSCIENCES AND HEALTH” (B&H) (M.G., 2013). The authors declare no conflicts of interest.
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Ranieri, E., Popescu, I., Gigante, M. (2014). CTL ELISPOT Assay. In: Ranieri, E. (eds) Cytotoxic T-Cells. Methods in Molecular Biology, vol 1186. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1158-5_6
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DOI: https://doi.org/10.1007/978-1-4939-1158-5_6
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