|Budget Amount *help
¥6,900,000 (Direct Cost : ¥6,900,000)
Fiscal Year 1997 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1996 : ¥4,200,000 (Direct Cost : ¥4,200,000)
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
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