Bensussan A, Mansur I G, Mallet V, Rodriguez A M, Girr M, Weiss E H, Brem G, Boumsell L, Gluckman E, Dausset J
Institut National de la Santé de la Recherche Médicale Unité 93, Hôpital Saint-Louis, Paris, France.
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10292-6. doi: 10.1073/pnas.92.22.10292.
A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.
通过用表达 HLA-G 和人β2-微球蛋白的转染鼠 L 细胞免疫 HLA-B27/人β2-微球蛋白双转基因小鼠，获得了一种单克隆抗 HLA-G 单克隆抗体（mAb）。这种单克隆抗体命名为 BFL.1，通过流式细胞术分析，它能特异性识别免疫的表达 HLA-G 的细胞，而不与亲本未转染的细胞或 HLA-B7 和 HLA-A3 转染的 L 细胞结合，这表明它能区分经典的 HLA-A 和 -B 以及非经典的 HLA-G I 类分子。通过 BFL.1 与许多已知表达经典 HLA I 类蛋白的人类细胞系无反应性，进一步评估了这一点。此外，我们还表明，BFL.1 单克隆抗体还标记天然表达 HLA-G 的 JEG-3 和 HLA-G 转染的 JAR 人绒毛膜癌细胞系以及早孕胎盘细胞滋养层细胞亚群。通过对生物素化膜裂解物进行免疫沉淀进行了进一步的生化研究：与单克隆 W6/32 单克隆抗体一样，BFL.1 在表达 HLA-G 的细胞系中免疫沉淀出一种 39 kDa 的蛋白质，其大小与预测的全长 HLA-G1 异构体相对应。然而，与能免疫沉淀经典和非经典 HLA I 类重链的 W6/32 不同，BFL.1 单克隆抗体不识别 Ia 类产物。这样一种单克隆抗体应该是分析各种正常和病理人类组织中 HLA-G 蛋白表达以及确定翻译后的 HLA-G 产物功能的有用工具。
