CTL ELISPOT Assay and T Cell Detection

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 of 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 be also 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 Milteny 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 will be described