Uchigata Y, Spitalnik S L, Tachiwaki O, Salata K F, Notkins A L
J Exp Med. 1987 Jan 1;165(1):124-39. doi: 10.1084/jem.165.1.124.
To investigate the autoimmune pathogenesis of spontaneously occurring diabetes mellitus in BB rats, spleen cells of newly diagnosed diabetic BB rats were fused with mouse myeloma cells. Hybridoma supernatants were screened for antibodies by indirect immunofluorescence and by 51Cr-release assays using the RINm5F rat insulinoma cell line. One clone, E5C2, produced an IgM kappa antibody that was cytotoxic for RINm5F cells, but not for other rat cell lines nor for primary rat islet cells. However, treatment of primary rat islet cells with neuraminidase exposed surface antigens and rendered the cells susceptible to complement-mediated lysis by antibody E5C2. Using immunostaining of glycolipids separated by thin-layer chromatography, hapten inhibition assays with defined carbohydrates, and Western blots, the antigens recognized by E5C2 on RINm5F cells were identified as glycoproteins with molecular weights of 60,000 and 68,000. The antibody recognizes a carbohydrate antigen containing the sequence Gal beta 1-4GlcNAc-R, which on RINm5F cells is predominantly hidden by covalently bound sialic acid. These studies raise the possibility that hidden antigenic determinants on islet cells exposed by a variety of means may be the target of autoimmune attack.
为了研究BB大鼠自发性糖尿病的自身免疫发病机制，将新诊断的糖尿病BB大鼠的脾细胞与小鼠骨髓瘤细胞进行融合。通过间接免疫荧光以及使用RINm5F大鼠胰岛素瘤细胞系的51Cr释放试验，对杂交瘤上清液中的抗体进行筛选。一个克隆E5C2产生了一种IgM κ抗体，该抗体对RINm5F细胞具有细胞毒性，但对其他大鼠细胞系和原代大鼠胰岛细胞无细胞毒性。然而，用神经氨酸酶处理原代大鼠胰岛细胞会暴露表面抗原，并使细胞易受抗体E5C2介导的补体溶解作用。通过对薄层层析分离的糖脂进行免疫染色、用特定碳水化合物进行半抗原抑制试验以及蛋白质免疫印迹法，确定了E5C2在RINm5F细胞上识别的抗原为分子量为60,000和68,000的糖蛋白。该抗体识别一种含有Galβ1-4GlcNAc-R序列的碳水化合物抗原，在RINm5F细胞上，该抗原主要被共价结合的唾液酸所掩盖。这些研究提出了一种可能性，即通过各种方式暴露的胰岛细胞上隐藏的抗原决定簇可能是自身免疫攻击的目标。
