Development of a panel of monoclonal antibodies against the mineralocorticoid receptor
Development of a panel of monoclonal antibodies against the mineralocorticoid receptor
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DOI：
10.1210/en.2005-0860
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发表时间：
2006-03-01
期刊：
影响因子：
4.8
通讯作者：
Gomez-Sanchez, EP
中科院分区：
文献类型：
--
作者：
Gomez-Sanchez, CE;de Rodriguez, AF;Gomez-Sanchez, EP
盐皮质激素受体(MR)结合盐皮质激素和糖皮质激素。它们在多种组织中表达，并调节不同的功能。与其他主要的类固醇受体相比，人们对MR的调节和功能知之甚少，尽管其重要性已经变得越来越明显。此类研究的一个重大障碍是缺乏特定的高亲和力MR抗体。我们已经针对10种不同的多肽结合物制备了单抗，其中6种来自N端(A/B区)，4种来自C端(类固醇结合域)，预计它们对MR的个别亲和力将取决于其构象，而构象又取决于受体在细胞内的位置和与其相关的蛋白质。用转增强型绿色荧光蛋白-大鼠MR基因的中国仓鼠卵巢细胞和去肾上腺大鼠海马胞液中的蛋白，用Western blotts方法分析了与同源肽ELISA高滴度的杂交瘤克隆。对肾脏、心脏、结肠和脑进行免疫组织化学染色。被证明对蛋白质印迹分析和免疫组织化学最有用的抗体包括针对氨基酸1-18、-82、79-97和365-381的抗体。同一细胞中胞浆和胞核的免疫反应强度因抗体的不同而不同，这表明某些受体表位或多或少地暴露于细胞内受体的位置。综上所述，描述了识别MR不同部分的几种抗体，这些抗体应该有助于研究这两类类固醇激素作用的重要媒介。
Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1-18, 64-82, 79-97, and 365-381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.