Habets W J, Berden J H, Hoch S O, Van Venrooij W J
Eur J Immunol. 1985 Oct;15(10):992-7. doi: 10.1002/eji.1830151006.
Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.
系统性自身免疫疾病患者的血清中常含有抗小核核糖核蛋白（snRNP）颗粒的抗体。抗Sm抗体可与整套U1、U2、U4、U5和U6（U1-U6）核糖核蛋白颗粒发生反应，而抗（U1）RNP血清则特异性识别含有U1 RNA的颗粒。在此，我们使用16份人抗Sm血清、15份人抗（U1）RNP血清以及两种小鼠单克隆抗体进行了半定量免疫印迹，以确定哪些与snRNA相关的蛋白质携带抗原决定簇。几乎每一份（15/16）人抗Sm血清都能识别存在于一条28 kDa（B/B'）蛋白双峰和一条16 kDa（D）多肽上的表位。9份抗（U1）RNP血清也能识别B/B'双峰，但在所有情况下，与一种或多种特异性与U1 RNA相关的70 kDa、A或C抗原的反应要强得多。通过从硝酸纤维素印迹上的各个抗原条带洗脱的亲和纯化抗体组分表明，抗Sm反应性多肽B/B'和D含有共同表位。我们还报告了一份人抗Sm血清对B/B'双峰具有排他性特异性的发现，以及一种仅识别D蛋白的小鼠单克隆抗Sm抗体，这表明这些抗原也携带独特表位。在免疫沉淀试验中，纯化的抗B/B'和抗D抗体与（U1-U6）核糖核蛋白反应，而纯化的抗70 kDa、抗A和抗C抗体仅沉淀U1核糖核蛋白颗粒。最后，我们使用生化细胞分级分离程序确定了Sm和U1核糖核蛋白抗原的亚细胞定位。部分70 kDa和B/B'抗原存在于一种对核酸酶和高盐具有抗性的核亚结构中，通常称为核基质，而A和D抗原可通过核糖核酸酶处理及随后的高盐提取从HeLa细胞核中完全提取出来。
