Radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma.

Abstract

Abstract For this sensitive RIA for 1 alpha,25-dihydroxyvitamin D, we used antibodies to 1 alpha,25-dihydroxycholecalciferol-3-hemisuccinate conjugated to bovine serum albumin, raised in rabbits. These antibodies show a high affinity for 1 alpha,25-dihydroxyvitamin D3 but cross react with other vitamin D metabolites as well. Extraction and liquid chromatography are required to isolate the 1 alpha,25-dihydroxyvitamin D from human serum or plasma with benzene (analytical recovery, 96%) and chromatographed by "high-performance" liquid chromatography on a silica column. The sensitivity of the assay is 2 pg/tube. The between-assay CV is 12.6% (means = 61.8 ng/L, n = 27) and the within-assay CV is 7.4% (means = 61.8 ng/L, n = 15). The reference interval, obtained from data on serum of healthy persons, is 51.8 +/- 15.1 ng/L (means +/- SD, n = 91), with no sex-related difference. Patients with chronic renal failure have lower concentrations in serum, and patients suffering from calcium nephrolithiasis have increased concentrations. Our assay offers the advantage of a relatively short prepurification procedure and the production of large amounts of stable gamma-globulins that can be used for several years without loss of binding properties.