A selective and simplified radioimmunoassay of 25-hydroxyvitamin D3

A simple, non-chromatographic and selective assay for determination of 25OHD3 in serum is described. The serum sample is deproteinated with acetonitrile and the supernatant purified over a small prepacked cartridge, Sep-pak. In the eluate from the cartridge, 25OHD3 is measured by radioimmunoassay. The selectivity of the assay for 25OHD3 is improved by adding vitamin D2 to the antiserum; the assay is thus capable of measuring 25OHD3 in serum samples containing up to a ratio of 25OHD2/25OHD3 equal to 150. The simplicity, speed and the small amount of sample needed (0.5 ml) make this method suitable for use in a routine clinical laboratory. Because of its selectivity for 25OHD3 the assay is a necessary prerequisite in the further investigation of metabolic discrimination between vitamins D2 and D3.