Dussossoy D, Carayon P, Feraut D, Belugou S, Combes T, Canat X, Vidal H, Casellas P
Immunology Department, Sanofi Recherche, Montpellier, France.
Cytometry. 1996 May 1;24(1):39-48. doi: 10.1002/(SICI)1097-0320(19960501)24:1<39::AID-CYTO5>3.0.CO;2-D.
Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes from PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab.
根据从克隆的人外周型苯二氮䓬受体（PBR）基因推导的氨基酸序列，制备了针对该受体C末端片段的单克隆抗体（Mab 8D7）。针对纯化的PBR进行的免疫印迹实验表明，该抗肽抗体在变性条件下识别PBR的相应氨基酸序列。当用来自PBR转染酵母或THP1和U937细胞的线粒体膜进行免疫印迹分析时，在18 kDa处观察到高水平的免疫反应性，这是从cDNA推导的PBR分子量，证实了该抗体对该受体的特异性。此外，用完整线粒体进行的结合实验表明，该免疫原性序列可被抗体识别，这表明PBR的C末端片段面向胞质溶胶。使用该单克隆抗体，我们开发了一种能够精确量化每个细胞中PBR密度的技术。此外，通过流式细胞术分析和共聚焦显微镜对显示不同水平PBR的细胞系进行的细胞定位研究表明，Mab 8D7与抗线粒体单克隆抗体完全共定位。
