Development of a sensitive analyzer for multiple immune function.

产生可溶性因子的单细胞的直接计数方法，例如ELISPOT测定，经常用于各种领域的研究中。然而，对测定中形成的斑点进行计数需要花费大量的时间和精力，而且是主观的。因此，我们试图通过使用图像分析仪来开发产生可溶性因子的单细胞自动计数器。首先，我们建立了产生抗甲状腺过氧化物酶自身抗体的淋巴细胞ELISPOT检测方法，并通过图像分析仪的数据处理使形成的斑点更加清晰。此外，我们通过从ELISPOT检测的染色中减去非特异性染色来减少微孔板孔中的硝酸纤维素膜的背景染色。不幸的是，获得具有不同大小和密度的斑点的总和的试验失败了，但是ELISPOT测定中形成的斑点的数量与测定中威尔斯孔的上清液中抗甲状腺过氧化物酶自身抗体的浓度相关。由于斑点的荧光强度较弱，无法建立淋巴细胞产生细胞因子的荧光SPOT法。我们建立了一个ELISPOT检测淋巴细胞产生可溶性膜蛋白（sCD 4或sCD 8）自发。sCD 8-产生细胞的数量在妊娠期间减少，但sCD 4-产生细胞的数量没有减少。本研究为研制单细胞免疫因子自动分析仪开辟了一条道路。

A direct counting method of single cells producing soluble factors, such as an ELISPOT assay, is frequently used in research of various fields. However, it takes a lot of time and effort to count spots formed in the assay, and furthermore subjective. Therefore, we tried to develop an automatic counter of single cells producing soluble factors by using an image analyzer. First, we set up an ELISPOT assay for lymphocytes producing anti-thyroid peroxidase autoantibodies, and make the formed spots clearer by data treatment of the image analyzer. Furthermore, we decreased the background staining of nitrocellulose membrane in the well of microplates by subtract the non-specific staining from the staining of the ELISPOT assay. Unfortunately, the trial to obtain the sum of spots with various sizes and density were failed, but the number of spots formed in the ELISPOT assay was correlated with the concentrations of anti-thyroid peroxidase autoantibodies in the supernatants of the wells in the assays. We could not set up the fluorescence SPOT assay for lymphocytes producing cytokines, because of the weak fluorescence intensity of spots. We set up an ELISPOT assay for lymphocytes producing soluble membrane proteins (sCD4 or sCD8) spontaneously. The number of sCD8-producing cells decreased during pregnancy, but that of sCD4-producing cells did not. In this study, we opened a way of developing an automatic analyzer for single immune cells producing soluble factors.