Van Leuven F, Marynen P, Cassiman J J, Van den Berghe H
Center for Human Genetics, University of Leuven, Belgium.
J Biochem. 1987 May;101(5):1181-9. doi: 10.1093/oxfordjournals.jbchem.a121982.
Mice were immunized with the isolated C-terminal heat-induced fragment of human alpha 2-macroglobulin (alpha 2M) and the spleens were used to prepare hybridomas. A monoclonal antibody (Mab) designated F17D5 was selected for further characterization. The epitope defined by Mab F17D5 was not expressed on alpha 2M, on alpha 2M-methylamine, or on alpha 2M-proteinase complexes. On the other hand, the antibody reacted avidly with denatured human alpha 2M and with some types of alpha 2M from other species, including mouse, on nitrocellulose-immunoblotting. The epitope of Mab F17D5 was mapped to less than 250 residues C-terminal of the internal thiolester of human alpha 2M. This was based on CNBr fragmentation of the 60 kDa C-terminal heat-fragment and on peptide mapping of alpha 2M, derivatized at the internal thiolester GLX-residue with 125I-labeled histamine. Murine alpha 2M was confirmed to contain two types of subunit: a 185 kDa subunit and a combination of 165 kDa and 35 kDa polypeptides. By partial disulfide bond reduction, heat-fragmentation and immunoblotting with Mab F17D5, the structure of murine alpha 2M was compared to that of human alpha 2M. The F17D5-epitope was mapped to a 30 kDa heat-induced fragment, which was released by denaturation without reduction. This fragment contained an intrachain disulfide bridge. By analogy with human alpha 2M, the 35 kDa subunit would be located at the C-terminal end of murine alpha 2M, disulfide-bonded to the major 165 kDa subunit.
用分离出的人α2-巨球蛋白（α2M）的C末端热诱导片段对小鼠进行免疫，然后用这些小鼠的脾脏制备杂交瘤。选择了一种名为F17D5的单克隆抗体（Mab）进行进一步鉴定。Mab F17D5所定义的表位在α2M、α2M-甲胺或α2M-蛋白酶复合物上均未表达。另一方面，在硝酸纤维素免疫印迹中，该抗体与变性的人α2M以及来自其他物种（包括小鼠）的某些类型的α2M发生强烈反应。Mab F17D5的表位被定位到人α2M内部硫酯C末端少于250个残基处。这是基于60 kDa C末端热片段的溴化氰裂解以及对α2M的肽图谱分析，α2M在内部硫酯GLX残基处用125I标记的组胺进行了衍生化。证实小鼠α2M含有两种亚基：一种185 kDa的亚基以及165 kDa和35 kDa多肽的组合。通过部分二硫键还原、热裂解以及用Mab F17D5进行免疫印迹，将小鼠α2M的结构与人α2M的结构进行了比较。F17D5表位被定位到一个30 kDa的热诱导片段，该片段在未还原的情况下通过变性释放。这个片段含有链内二硫键。与人α2M类似，35 kDa亚基将位于小鼠α2M的C末端，通过二硫键与主要的165 kDa亚基相连。
