• Elena RanieriEmail author
  • Iulia Popescu
  • Margherita Gigante
Part of the Methods in Molecular Biology book series (MIMB, volume 1186)


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.

Key words

Elispot T cell Cytokines Cancer 



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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Elena Ranieri
    • 1
    Email author
  • Iulia Popescu
    • 2
  • Margherita Gigante
    • 3
  1. 1.Department of Surgical and Medical Sciences, School of MedicineUniversity of FoggiaFoggiaItaly
  2. 2.Division of Pulmonary Allergy and Critical Care Medicine, Department of MedicineUniversity of PittsburghPittsburghUSA
  3. 3.Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, PoliclinicoUniversity of Bari “A. Moro”BariItaly

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