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

在本研究中，我们试图建立一种有用的方法来检测系统性红斑狼疮或混合性结缔组织病患者外周血单个核细胞中发现的U1-68k自身免疫原去磷酸化形式。作为传统2D-Western印迹方法的替代，我们检查了三种方法：(1)免疫学方法，(2)PHOS-Tag电泳法，(3)等电聚焦电泳法-Western印迹法。结果发现，等电聚焦电泳-蛋白质印迹法最适合于U1-68k去磷酸化形式的检测。此外，我们还发现，用含有十二烷基硫酸钠的细胞裂解缓冲液制备的全细胞提取液可以用于检测U1-68k。综上所述，等电聚焦电泳-Western印迹结合含十二烷基硫酸钠的细胞裂解缓冲液提取全细胞可将分析时间从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.