Okamoto Y, Abe T, Niwa T, Mizuhashi S, Nishida M
Department of Clinical Pharmacy, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
Immunopharmacology. 1998 May;39(2):107-16. doi: 10.1016/s0162-3109(98)00007-1.
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/or interleukin-4 (IL-4) at a single cell level, a dual color ELISPOT assay has been developed. In the present system, the red spots corresponding to IL-2-secreting cells (T helper type 1, Th1 cells) were developed with a horseradish peroxidase and the amino ethyl carbazole (AEC)/H2O2. The light 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 mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (T helper type 0, Th0 cells) were developed with both substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in crude spleen cell preparation or purified CD4 fraction. This procedure provides a useful tool for quantitatively analyzing micro-levels of dynamic immune responses.
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。为了在单细胞水平上同时区分产生白细胞介素-2（IL-2）和/或白细胞介素-4（IL-4）的单个细胞，已开发出双色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）相对应的浅蓝色斑点用碱性磷酸酶和Vector blue显色。与两种细胞因子分泌细胞（0型辅助性T细胞，Th0细胞）相对应的混合颜色（靛蓝）斑点用两种底物显色。利用该系统，我们可以在粗制脾细胞制剂或纯化的CD4组分中同时检测分泌IL-2和/或IL-4的细胞。该方法为定量分析微观水平的动态免疫反应提供了一个有用的工具。
