Development of a sensitive analyzer for multiple immune function.

单细胞产生可溶性因子的直接计数方法，如ELISPOT法，经常被用于各种领域的研究。然而，对化验中形成的斑点进行计数需要花费大量的时间和精力，而且还具有主观性。因此，我们试图利用图像分析仪开发一种单细胞产生可溶性因子的自动计数器。首先，我们建立了淋巴细胞产生抗甲状腺过氧化物酶自身抗体的ELISPOT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，通过从ELISPOT染色中减去非特异性染色，我们减少了硝酸纤维素膜在微孔板中的背景染色。不幸的是，获得不同大小和密度的斑点总数的尝试失败了，但ELISPOT试验中形成的斑点数量与检测孔上清中抗甲状腺过氧化物酶自身抗体的浓度有关。我们无法建立产生细胞因子的淋巴细胞的荧光斑点试验，因为斑点的荧光强度很弱。我们建立了淋巴细胞自发产生可溶性膜蛋白(sCD4或sCD8)的ELISPOT方法。在妊娠期间，产生sCD8的细胞数量减少，但产生sCD4的细胞数量没有减少。在这项研究中，我们开辟了一种用于单个免疫细胞产生可溶性因子的自动分析仪的开发方法。

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.