title: Quantification of antigen specific CD8+ T cells using an ELISPOT assay.
abstract: An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.
authors.affiliation: Department of Medical and Molecular Parasitology, New York University Medical Center, NY 10010, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Miyahira
authors.forename: K
authors.initials: K
authors.lastname: Murata
authors.forename: D
authors.initials: D
authors.lastname: Rodriguez
authors.forename: J R
authors.initials: JR
authors.lastname: Rodriguez
authors.forename: M
authors.initials: M
authors.lastname: Esteban
authors.forename: M M
authors.initials: MM
authors.lastname: Rodrigues
authors.forename: F
authors.initials: F
authors.lastname: Zavala
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000032:analysis; D007371:Interferon-gamma / Q000032:analysis; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D008288:Malaria / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010967:Plasmodium yoelii / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D000953:Antigens, Protozoan; D000939:Epitopes; D007371:Interferon-gamma