title: CTL ELISPOT assay.
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.
authors.affiliation: Department of Surgical and Medical Sciences, School of Medicine, University of Foggia, Ospedali Riuniti, Viale Luigi Pinto, 1, Foggia, 71122, Italy, elena.ranieri@unifg.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Ranieri
authors.forename: Iulia
authors.initials: I
authors.lastname: Popescu
authors.forename: Margherita
authors.initials: M
authors.lastname: Gigante
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D053804:Granzymes / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000276:immunology; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D009369:Neoplasms / Q000175:diagnosis* / Q000276:immunology; D009975:Orthomyxoviridae / Q000276:immunology; D054353:Perforin / Q000032:analysis* / Q000378:metabolism; D013194:Staining and Labeling; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018984:Epitopes, T-Lymphocyte; D054353:Perforin; D007371:Interferon-gamma; D053804:Granzymes