Slobbe R L, Pruijn G J, Damen W G, van der Kemp J W, van Venrooij W J
Department of Biochemistry, University of Nijmegen, The Netherlands.
Clin Exp Immunol. 1991 Oct;86(1):99-105. doi: 10.1111/j.1365-2249.1991.tb05780.x.
The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren's syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren's syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren's syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.
通过将凝胶中的交联水平改变为丙烯酰胺/双丙烯酰胺比例为19:1，免疫印迹法同时检测抗La、抗60-kD Ro和抗52-kD Ro抗体的效果得到了极大改善。使用这种方法分析了许多系统性红斑狼疮（SLE）和干燥综合征患者的血清，结果发现，仅有抗52-kD Ro蛋白抗体而无抗60-kD Ro抗体的情况仅限于干燥综合征患者（26例中有9例），而仅有抗60-kD Ro蛋白抗体且无抗52-kD Ro抗体污染的情况仅在SLE患者中发现（38例中有8例）。此外，在干燥综合征患者血清中，抗Ro抗体仅与抗La抗体同时出现（26例中有20例），而在SLE患者血清中，可检测到无抗La特异性的抗Ro抗体（38例中有4例）。双重免疫荧光显微镜检查显示，52-kD Ro和60-kD Ro蛋白在细胞质和细胞核中均共定位，而用单特异性抗52-kD Ro和抗60-kD Ro血清对[32P]标记的HeLa细胞提取物进行免疫沉淀表明，这两种蛋白均与Ro RNA相关。这些数据表明，52-kD和60-kD Ro蛋白存在于同一核糖核蛋白复合物中。为了研究52-kD Ro、60-kD Ro和La蛋白的进化保守性，使用单特异性人抗体通过蛋白质印迹法分析了来自各种哺乳动物物种的细胞系提取物。与在进化中高度保守的60-kD Ro和La抗原不同，仅通过这种免疫方法才能在灵长类细胞中检测到52-kD Ro抗原。
