Development of assay method of serum 1,25-dihydroxyvitamin D concentration without radioisotope

为了便于骨质疏松症的诊断和预防，需要精确测定血清1，25-二羟基维生素D(1，25(OH)2D)浓度。目前的检测方法是用从鸡肠或牛胸腺中部分纯化的维生素D受体(VDR)，以[~3H]1，25(OH)2D3为标准。这些方法的缺点是由于放射性同位素的使用和其他维生素D代谢物的干扰而受到实验室的限制。因此，需要对样品进行多次前处理，从而浪费时间和成本。在此基础上，利用生物素-亲和素结合酶受体测定法，探索了一种简便、廉价的检测方法。首先，建立了大量表达人维生素D受体融合谷胱甘肽S-转移酶的大肠杆菌系统。该VDR与[3H]1，25(OH)2D3具有稳定的结合能力。对VDR进行了纯化，并研究了它们与[~3H]1，25(OH)2D3的结合能力。The Sensitive…在25(OH)D中，除1，25(OH)2D外，维生素D代谢物的作用略有观察，但敏感性高于牛胸腺。这些结果表明，在大肠杆菌中表达的VDR有助于改进人血清1，25(OH)2D浓度的测定。下一步，我们制备了识别1，25(OH)2D的抗体用于酶受体检测。结果获得了可识别VDR的高滴度抗体用于蛋白质印迹分析，尽管这种稀释10倍的抗体抑制了1，25(OH)2D与VDR的结合。这一结果表明该抗体不能用于敏感的酶受体检测。虽然许多抗体都是干扰结合的，但还是制备了许多抗体。从这些研究中可以看出，克服了许多步骤，取得了很大的进展。进一步阐明了人VDR的新型抗体和生物素标记的1，25(OH)2D3的合成是建立无放射性同位素的人血清1，25(OH)2D浓度测定方法的基础。较少

For the easy diagnosis and prophylaxis of osteoporosis, the precise measurement of serun 1,25-dihydroxyvitamin D (1,25 (OH) 2D) concentration is required. The assays at present were carried out by using the vitamin D receptor (VDR) partially purified from chicken intestine or cow thymus and [3H] 1,25 (OH) 2D3 as standard. The disadvantages of these methods are the limitations of laboratory because of radioisotome use and other vitamin D metabolites interfere these. Therefore, several pretreatment of sample are required and consequently waste time and cost. From these, the development of easy and cheep assay method was investigated using enzyme receptor assay applying biotin-avidin procedure in this research project.First, the E.coli system expressing large amount of human VDR fusing glutathione S-transferase was established. This VDR showed constant binding ability with [3H] 1,25 (OH) 2D3. The VDR was purified and their binding ability to [3H] 1,25 (OH) 2D3 was studied. The sensitivity … More was same as that using cow thymus VDR.The effect of vitamin D metabolites besides 1,25 (OH) 2D was slightly observed in 25 (OH) D,although better sensitivity was shown than that using cow thymus. From these results, it is concluded VDR expressed in E.coli was useful for the improved measurement of human serum 1,25 (OH) 2D concentration.In next step, antibody for human VDR was prepared to recognize 1,25 (OH) 2D for enzyme receptor assay. As a result, high titer antibody that could recognized VDR for western blot analysis was obtained, although 1,25 (OH) 2D binding to VDR was inhibited by this 10-fold diluted antibody. This result indicated that the antibody could not utilize for sensitive enzyme receptor assay. Many antibodies were prepared althoug they were all interfered the binding.It is condluded from these investigations using this Grant-in-Aid for Scientific Research that many steps were overcome and great progress were made. Furthermore, it is clarified that the novel antibody for human VDR and the synthesis of biotine labeled 1,25 (OH) 2D3 will be requred for the development of human serum 1,25 (OH) 2D concentration assay method without radioisotope. Less