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

我们试图开发一种高灵敏度的检测方法，利用自动计数系统检测产生特定抗体或细胞因子的细胞。首先，我们开发了一种ELISPOT方法，用于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，该检测方法的敏感性高于放射免疫检测方法。其次，我们开发了一种使用彩色图像分析仪对ELISPOT分析中每个孔底形成的斑点进行自动计数的系统。在该系统中，每个井底的场景包括在金属显微镜下放大的斑点，放大后的场景由彩色电视摄像机拍摄，并由高性能彩色图像分析仪进行分析。通过用孔扫描仪在金属显微镜上移动板来依次分析板的每一个孔。在高性能分析仪中，只对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井中颜色显影的程度。因此，我们从测试井中减去了对照井中的颜色显影程度。使用该系统，我们发现分析仪计数的斑点数与人工计数的斑点数之间存在显著的相关性。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检测，但很难计算产生IL-2或干扰素-γ的细胞在ELISPOT检测中形成的苍白斑点。

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.