A highly sensitive method for large-scale measurements of 1,25-dihydroxyvitamin D

A quantitative method for measuring 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was developed utilizing a luciferase reporter gene under the control of the highly inducible 25-hydroxyvitamin D3 24-hydroxylase promoter in a stably transfected cell line. Transient transfections with constructs containing the 24-hydroxylase gene promoter 5' to a luciferase 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 transfected with the plasmid, gave a 60-fold stimulation with 10(-10) M 1,25-(OH)2D3. A standard curve was constructed showing a large range of response to 1,25-(OH)2D3 (1 pg to 1 ng). The assay was adapted to microtiter plates, which permits a large number of samples to be assayed simultaneously. Other metabolites of vitamin D and analogs such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1 alpha-hydroxyvitamin D3 have negligible effects on the detection of 1,25-(OH)2D3, thus eliminating the need for purification of sample. The sensitivity of the method permitted the use of 100 microliters of serum with excellent results. Comparison of this method with a commercially available assay demonstrates that it gives higher sensitivity, simpler manipulations, and comparable results.