Labeta M O, Fernandez N, Festenstein H
Department of Immunology, London Hospital Medical College, U.K.
J Immunol Methods. 1988 Aug 9;112(1):133-8. doi: 10.1016/0022-1759(88)90043-9.
We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.
我们分析了三种去污剂对细胞膜组织相容性糖蛋白的差异增溶作用。使用了两种广泛用于提取MHC蛋白的非离子去污剂（Nonidet P - 40和Triton X - 100）以及一种两性离子去污剂（CHAPS），它是胆酸的磺基甜菜碱衍生物。以AKR（H - 2k）来源的自发白血病细胞系——424——作为实验模型。在这个肿瘤细胞系中表达了一种类I抗原，但通过细胞结合放射免疫测定或从NP - 40或Triton X - 100增溶的糖蛋白中进行免疫沉淀无法直接检测到。然而，在用CHAPS增溶的424糖蛋白与34.5.8抗H - 2Dd单克隆抗体进行免疫沉淀后，通过SDS - PAGE可见与经典H - 2 I类模式一致的46 kDa和12 kDa条带。H - 2Dd反应性分子似乎与至少一种同基因I类特异性（H - 2Kk，H - 2Dk）相关，并且无法与特异性抗H - 2Dd单克隆抗体反应。去污剂NP - 40和Triton X - 100在破坏蛋白质 - 蛋白质相互作用方面似乎不如CHAPS有效。CHAPS的这一特性使得新型抗原能够充分增溶并直接检测。这项研究的结果表明，非变性两性离子去污剂的交替使用可能有助于检测和表征肿瘤及正常细胞中与MHC相关的膜结合蛋白。
