Establishment of maker for early diagnosis of systemic autoimmune diseases based on de-phosphorylation of autoimmogen U1-68k

在这项研究中，我们试图建立一种有用的方法来检测一种去磷酸化形式的U1-68k自身免疫原，发现它在系统性红斑狼疮或混合性结缔组织疾病患者的外周血单个核细胞中增加。作为传统二维免疫印迹方法的替代，我们研究了三种方法；(1)免疫学方法，(2)光标记电泳，(3)等电聚焦电泳-western blot。因此，我们发现等电聚焦电泳-western blot方法最适合检测U1-68k的去磷酸化形式。此外，我们发现含有SDS的细胞裂解缓冲液制备的全细胞提取物可用于检测U1-68k。综上所述，等电聚焦电泳-western blot与含细胞裂解缓冲液的全细胞提取相结合，可将分析时间从5天缩短至2.5天。

In this study, we tried to establish an useful method for detecting a de-phosphorilated form of U1-68k autoimmunogen, which was found to be increased in peripheral blood mononuclear cells of patients of systemic lupus erythematosus or Mixed connective tissue diseases. As a substitution of a conventional 2D-western blot method, we examined three methods; (1) an immunological method, (2) a phos-tag electrophorsis, and (3)an isoelectric focusing electrophoresis-western blot. As a result, we found that the isoelectric focusing electrophoresis-western blot method is most suitable for the detection of the de-phosphorilated form of U1-68k. Moreover, we found that whole cell extract prepared by cell lysis buffer containing SDS can be used for detecting U1-68k. In conclusion, combination of isoelectric focusing electrophoresis-western blot and whole cell extract by SDS-containing cell lysis buffer can shorten analsys time from 5 days to 2.5 days.