Miyahira Y, Murata K, Rodriguez D, Rodriguez J R, Esteban M, Rodrigues M M, Zavala F
Department of Medical and Molecular Parasitology, New York University Medical Center, NY 10010, USA.
J Immunol Methods. 1995 Apr 12;181(1):45-54. doi: 10.1016/0022-1759(94)00327-s.
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.
利用针对啮齿动物疟疾抗原表位SYVPSAEQI的克隆鼠CD8+ T细胞，对检测和确定抗原特异性CD8+ T细胞数量的ELISPOT检测法进行了标准化。该检测法基于单细胞在用抗原刺激后分泌的γ干扰素的检测。干扰素分泌通过使用酶标抗γ干扰素单克隆抗体呈现为斑点来可视化。使用已知数量的克隆鼠CD8+ T细胞确定该检测法可检测到这些CD8+ T细胞的80 - 95%。确定了刺激CD8+ T细胞的最佳培养条件，并确定了进行该检测所需的抗原浓度、抗原呈递细胞数量和生长因子补充剂。这种ELISPOT检测法可用于免疫小鼠的脾细胞，能提供混合淋巴细胞群体中存在的抗原特异性CD8+ T细胞的精确数量。该方法比铬-51释放检测法更灵敏，且比传统的前体细胞频率分析简单得多，可在36 - 48小时内提供抗原特异性CD8+ T细胞的数量。
