Okamoto Y, Gotoh Y, Shiraishi H, Nishida M
Division of Health Care Pharmacy, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan.
Int Immunopharmacol. 2004 Jan;4(1):149-56. doi: 10.1016/j.intimp.2003.08.008.
The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies and cytokines. For simultaneous differentiation of individual cells producing interleukin-2 (IL-2) and interleukin-4 (IL-4) at a single cell level in human peripheral blood mononuclear cells (PBMCs), a human dual-color ELISPOT assay has been optimized. In the present system, the red spots corresponding to IL-2-secreting cells (T helper type 1, Th1, cells) were developed with horseradish peroxidase and the amino ethyl carbazole (AEC)/H2O2. The blue spots corresponding to IL-4-secreting cells (T helper type 2, Th2, cells) were developed with an alkaline phosphatase and the Vector blue. The usefulness of the assay method was tested. With this system, we could detect the IL-2- and IL-4-secreting cells simultaneously in human PBMCs of a juvenile rheumatoid arthritis (JRA) patient. This procedure provides useful information on clinical immune disorders.
酶联免疫斑点（ELISPOT）测定法是一种用于计数分泌抗体和细胞因子的单个细胞的有效技术。为了在人外周血单个核细胞（PBMC）中在单细胞水平上同时区分产生白细胞介素-2（IL-2）和白细胞介素-4（IL-4）的单个细胞，已优化了一种人双色ELISPOT测定法。在本系统中，与分泌IL-2的细胞（1型辅助性T细胞，Th1细胞）相对应的红色斑点用辣根过氧化物酶和氨基乙基咔唑（AEC）/H2O2显色。与分泌IL-4的细胞（2型辅助性T细胞，Th2细胞）相对应的蓝色斑点用碱性磷酸酶和Vector blue显色。测试了该测定方法的实用性。使用该系统，我们可以在一名幼年类风湿性关节炎（JRA）患者的人PBMC中同时检测到分泌IL-2和IL-4的细胞。该程序为临床免疫紊乱提供了有用的信息。
