Sandgren M E, Deluca H F
Department of Biochemistry, University of Wisconsin-Madison 53706.
Anal Biochem. 1989 Nov 15;183(1):57-63. doi: 10.1016/0003-2697(89)90171-1.
A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.
已开发出一种不依赖配体的定量测定方法，用于测量1,25 - 二羟基维生素D受体。该方法以从猪小肠中纯化的受体作为标准品，并使用两种针对受体上两个不同表位的高亲和力单克隆抗体。在此测定中，将固定量的125I标记抗体与固定量的与生物素相连的第二抗受体抗体以及不断增加量的纯化受体蛋白或样品一起孵育。然后可用抗生物素蛋白 - 琼脂糖珠免疫沉淀抗体 - 受体复合物并进行计数。该方法具有高度可重复性，能够在粗提物中检测到150 pg的1,25 - 二羟基维生素D3受体，批内和批间变异系数分别为8.6%和18.2%。所使用的单克隆抗体能够识别包括人类在内的几种不同物种的天然和变性受体。这种免疫放射测定法对于受体调节、占有率、分布和周转的研究应是有用的。
