小倉 勝 ヤマサ醤油, 診断薬部, 部統括
HIGASHI Tatsuya Fac.Pharm.Sci., Kanazawa Univ., 薬学部, 助手 (90272963)
KOBAYASHI Norihiro Fac.Pharm.Sci., Kanazawa Univ., 薬学部, 助手 (90205477)
OGURA Masaru Dep.Diagnostic Reagent, Yamasa Co., Head
1 alpha, 25-Dihydroxyvitamin D_3 [1,25 (OH) _2D_3] is the hormonally active form of vitamin D_3 which plays an important role in regulating calcium and phosphorus metabolism and bone resorption.. The measurement of 1,25 (OH) _2D_3 levels in human serum/plasma is of great value for clinical diagnosis, such as osteoporosis. Conventionally, receptor assays (RRAs) based on vitamin D receptor have been the main method for determining the 1,25 (OH) _2D_3 levels. However, these methods required exhaustive purification steps of the biological samples including preparative HPLC.
A selective and simple immunoaffinity chromatography (IAC) has been developed and found to be useful pretreatment tool for determining 1,25 (OH) _2D_3 in human plasma. A reasonably designed haptenic derivatives, 11 alpha-hemiglutaryloxy-1,25 (OH) _2D_3, was linked to bovine serum albumin, and rabbits were immunized with the conjugate. The resulting polyclonal antibodies were specific to 1,25 (OH) _2D_3, recognizing both the A-ring and the side-chain structures. The antibodies were then immobilized on agarose gel and used as an IAC which enabled a simple RRA assay for human plasma 1,25 (OH) _2D_3. The satisfactory monoclonal antibodies have also been produced against the haptenic derivatives, which will be applicable for developing IAC.
The determination methods for the conjugated metabolites of vitamin D using HPLC have also been developed.