A HIGHLY SENSITIVE METHOD FOR LARGE-SCALE MEASUREMENTS OF 1,25-DIHYDROXYVITAMIN-D

A quantitative method for measuring 1,25-dihydroxyvitamin D-3 (1,25-(O H)(2)D-3) was developed utilizing a luciferase reporter gene under the control of the highly inducible 25-hydroxyvitamin D-3 24-hydroxylase promoter in a stably transfected cell line. Transient transfections wi th constructs containing the 24-hydroxylase gene promoter 5' to a luci ferase reporter were first performed in cell lines with high levels of vitamin D receptor, i.e., the rat osteosarcoma (ROS 17/2.8) and human breast cancer (T-47D) cell lines. ROS 17/2.8 cells, stably transfecte d with the plasmid, gave a 60-fold stimulation with 10(-10) M 1,25-(OH )(2)D-3. A standard curve was constructed showing a large range of res ponse to 1,25-(OH)(2)D-3 (1 pg to 1 ng). The assay was adapted to micr otiter plates, which permits a large number of samples to be assayed s imultaneously. Other metabolites of vitamin D and analogs such as 25-h ydroxyvitamin D-3, 24,25-dihydroxyvitamin D-3, and 1 alpha-hydroxyvita min D-3 have negligible effects on the detection of 1,25-(OH)(2)D-3, t hus eliminating the need for purification of sample. The sensitivity o f the method permitted the use of 100 mu l of serum with excellent res ults. Comparison of this method with a commercially available assay de monstrates that it gives higher sensitivity, simpler manipulations, an d comparable results. (C) 1998 Academic Press.