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Development of an automatic high-sensitive assay system for cells producing a specific antibody or a cytokine.

开发用于产生特定抗体或细胞因子的细胞的自动高灵敏度检测系统。

基本信息

批准号：
02557108
负责人：
IWATANI Yoshinori
金额：
$ 7.94万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557108/
关键词：
Specific antibody-producing cells Cytokine-producing cells ELISPOT assay Automation Standardization

项目摘要

We tried to develop a high-sensitive assay for detection of cells producing a specific antibody or a cytokine with the automatic counting system. First, we developed an ELISPOT assay for cells producing thyroid-specific autoantibodies. The sensitivity of this assay was higher than that of a radioimmunoassay in terms of the detection of thyroid-autoantibody production. Second, we developed an automatic counting system for spots formed in the bottom of each well in an ELISPOT assay using an analyzer for color pictures. In this system, a scene of the bottom of each well including spots magnified in a metal microscope, and the magnified scene was shot by a color TV camera and analyzed by high-performance analyzer for color pictures. Each well of a plate was analyzed in turn by moving the plate on the metal microscope with a well scanner. In the high-performance analyzer, only spots with given ranges of size and depth were counted, but the counting number of spots depended on the degree of color development in the well. Therefore, we subtracted the degree of color development in a control well from that in a test well. Using this system, we found a significant correlation between the spot numbers counted by the analyzer and by manual counting. Furthermore, this system can be used for ELISPOT assays for cells producing various immunoglobulin classes of thyroid autoantibodies, but it was difficult to count the pale spots formed in a ELISA assay for cells producing IL-2 or IFN-gamma.

我们试图开发一种高灵敏度的自动计数系统检测产生特异性抗体或细胞因子的细胞的方法。首先，我们开发了一种ELISPOT检测产生甲状腺特异性自身抗体的细胞。在检测甲状腺自身抗体产生方面，该测定法的灵敏度高于放射免疫测定法。其次，我们开发了一个自动计数系统，用于ELISPOT分析中每个孔底部形成的斑点，使用彩色图片分析仪。在该系统中，每个孔的底部的场景包括在金属显微镜中放大的斑点，并且放大的场景由彩色电视摄像机拍摄并由高性能分析仪分析彩色图像。通过用孔扫描仪在金属显微镜上移动板，依次分析板的每个孔。在高性能分析仪中，仅对具有给定大小和深度范围的斑点进行计数，但斑点的计数数量取决于孔中显色的程度。因此，我们从测试孔中减去对照孔中的显色程度。使用该系统，我们发现了显着的相关性之间的斑点数量计数的分析仪和手动计数。此外，该系统可用于产生各种免疫球蛋白类甲状腺自身抗体的细胞的ELISPOT测定，但难以计数在产生IL-2或IFN-γ的细胞的ELISA测定中形成的苍白斑点。