MONOCLONAL-ANTIBODIES TO HUMAN PROGESTERONE-RECEPTOR - CHARACTERIZATION BY BIOCHEMICAL AND IMMUNOHISTOCHEMICAL TECHNIQUES
MONOCLONAL-ANTIBODIES TO HUMAN PROGESTERONE-RECEPTOR - CHARACTERIZATION BY BIOCHEMICAL AND IMMUNOHISTOCHEMICAL TECHNIQUES
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DOI：
10.1210/endo-121-3-1123
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发表时间：
1987-09-01
期刊：
影响因子：
4.8
通讯作者：
SATYASWAROOP, PG
中科院分区：
文献类型：
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作者：
CLARKE, CL;ZAINO, RJ;SATYASWAROOP, PG
在裸鼠体内生长的人子宫内膜癌(ENCA 101)的孕激素受体(PR)由两种激素结合蛋白组成，分子质量约为116,000和85,000。为了制备抗该受体的单抗，从eNCA 101中部分纯化了PR，并将其用于免疫罗伯逊小鼠。将免疫小鼠脾与HL-1友好的骨髓瘤-653细胞融合，用固相斑点杂交和双抗体沉淀法筛选杂交瘤细胞。获得了7株稳定的杂交瘤细胞，命名为HPRA1-7。亚型分析表明，HPRA1和HPRA6为免疫球蛋白G2B，其余为免疫球蛋白G1。高盐蔗糖梯度超速离心法显示，7种抗体中有6种能使[3 H]孕激素标记的PR从eNCA 10 1移位，只有HPRA 4对此无效。ENCA 101胞浆和DEAE洗脱液的蛋白质印迹显示，HPRA 1、3、4、5和7对这两种PR蛋白具有相同的识别能力。HPRA 2主要识别116,000摩尔重量的PR蛋白；它识别较低摩尔重量的PR蛋白非常差，而HPRA 6仅识别116,000摩尔重量的PR蛋白。有趣的是，后者一直被检测为紧密迁移的三胞胎。HPRA 1-7在组织切片中表达PR仅限于靶组织的细胞核，且强度不同：HPRA 7>；3=5>；6=2>；1>；4.增生期子宫内膜腺核(3+)>；肌层细胞核(2-3+)>；子宫内膜间质细胞核(0-1+)。因此，我们制备了7种针对人PR的单抗，并证明了它们适合于用生化和免疫组织化学技术研究PR。
Progesterone receptor (PR) from a human endometrial carcinoma (EnCa 101) grown in nude mice consists of two hormone-binding proteins with mol wt around 116,000 and 85,000. To generate monoclonal antibodies against this receptor, PR was partially purified from EnCa 101 and used to immunize Robertsonian mice. Immune mouse spleens were fused with HL-1 Friendly myeloma-653 cells, and hybridomas were screened by solid phase dot-blot assay and double antibody precipitation. Seven stable hybridomas were obtained, designated hPRa 1-7. Subisotyping revealed that hPRa 1 and 6 were immunoglobulin G2b, while the remainder were immunoglobulin G1. Ultracentrifugation in high salt sucrose gradients showed that six of the seven antibodies effected a shift of [3H]progestin-labeled PR from EnCa 101; only hPRa 4 was ineffective in this regard. Protein blots of EnCa 101 cytosols and DEAE eluates revealed that hPRa 1, 3, 4, 5, and 7 recognized both PR proteins equally. hPRa 2 recognized principally the 116,000 mol wt PR protein; it recognized the lower mol wt PR protein very poorly if at all, whereas hPRa 6 recognized only the 116,000 mol wt protein. Interestingly, the latter was consistently detected as a closely migrating triplet. Immunolocalization of PR by hPRa 1-7 in tissue sections was confined to nuclei of target tissues and varied in intensity: hPRa 7 > 3 = 5 > 6 = 2 > 1 > 4. In proliferative phase uterus, the intensity of staining was ranked: endometrial gland nuclei (3+) > myometrial cell nuclei (2-3+) > endometrial stromal cell nuclei (0-1+). Thus, seven monoclonal antibodies directed against human PR have been prepared, and their suitability for the study of PR by biochemical and immunohistochemical techniques has been demonstrated.