Uhland-Smith A, Prahl J M, DeLuca H F
Department of Biochemistry, University of Wisconsin-Madison, USA.
J Bone Miner Res. 1996 Dec;11(12):1921-5. doi: 10.1002/jbmr.5650111213.
In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated. The receptor-antibody complex is detected with avidin-alkaline phosphatase and rho-nitrophenyl phosphate. The amount of receptor in each sample is determined by comparison with a standard curve made from purified receptor protein. This assay is highly sensitive, measuring as little as 2 fmol of receptor, and has an intra-assay coefficient of variation of 6.6% and an interassay coefficient of variation of 13.8%. The assay can be used to measure the receptor from mammalian and avian species and is independent of the presence of hormone. By eliminating the need for a radio-iodinated monoclonal antibody and incorporating the ease of a plate assay, we have a significantly improved method for measuring the vitamin D receptor protein. This paper also presents Western analysis of the antibodies used to demonstrate that they do not recognize other steroid hormone receptors.
在本文中，我们详细介绍了一种用于检测1,25 - 二羟基维生素D3受体蛋白的酶联免疫测定法。细胞和组织匀浆中的受体蛋白结合在针对该受体蛋白不同表位的两种单克隆抗体之间。第一种抗体结合在酶联免疫吸附测定（ELISA）板的孔中，第二种抗体用生物素进行了标记。受体 - 抗体复合物通过抗生物素蛋白 - 碱性磷酸酶和对硝基苯磷酸酯进行检测。通过与由纯化的受体蛋白制成的标准曲线进行比较，确定每个样品中受体的含量。该测定法高度灵敏，可检测低至2飞摩尔的受体，测定内变异系数为6.6%，测定间变异系数为13.8%。该测定法可用于检测来自哺乳动物和鸟类物种的受体，且不受激素存在的影响。通过无需放射性碘化单克隆抗体并采用简便的板测定法，我们拥有了一种显著改进的用于检测维生素D受体蛋白的方法。本文还展示了用于证明这些抗体不识别其他类固醇激素受体的蛋白质免疫印迹分析。
