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

无放射性同位素血清1,25-二羟基维生素D浓度测定方法的建立

基本信息

批准号：
07557321
负责人：
TAKEDA Eiji
金额：
$ 4.42万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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

目前，使用tamin d受体（VDR）进行了简单的诊断和预防性。方法是实验室使用的局限性，其他维生素D代谢物会干扰这些样品，因此需要进行严重的样品预处理，因此使用酶来调查了易于生物素的方法。首先，表达大量人类VDR的谷胱甘肽S-转移酶的E.COLI系统在[3H] 1,25（OH）2D3中建立了大量的E.COLI系统。研究了[3H] 25（OH）2d3的敏感性。从这些结果中，在E.Coli中表达的VDR从乳房中进行了，可用于改善人血清1,25（OH）2D浓度。受体测定。结果，高滴度的ant ant识别为印迹分析的VDR，Althooth 1,25（OH）是10倍稀释的抗体科学的研究是克服了许多步骤，并取得了巨大的进步，并将标记为1,25（OH）2D3的生物定为开发人类血清1,25（OH）AY方法