A sensitive and selective radioimmunoassay for serum 24,25-dihydroxycholecalciferol in man

We have developed a sensitive and selective radioimmunoassay for 24,25-dihydroxycholecalciferol (24,25(OH)2D3). Antisera with a high titre and affinity to 24,25(OH)2D3 were raised in rabbits immunized with a protein conjugate of the 3-hemisuccinate derivative of 24,25(OH)2D3. Serum samples were extracted by methanol/dichloromethane and the lipid extracts purified on Sephadex LH 20 and chromatographed by single step HPLC on a straight phase silica column. The radioimmunoassay is capable of measuring 24,25(OH)2D3 in the '24,25(OH)2D complex' isolated by HPLC and containing the comigrating metabolites: 24,25(OH)2D3, 24,25(OH)2D2, 25(OH)D3-26,23 lactone and 25,26(OH)2D2. The detection limit of 2 pg/assay tube is a marked improvement, compared to the competitive protein binding assay using rachitic rat serum. Measured by the radioimmunoassay 24,25(OH)2D3 ranged from 0.05 to 1.96 ng/ml with a mean of 0.85 ng/ml in 34 healthy adults. For comparison 24,25(OH)2D, measured simultaneously by competitive protein binding with rachitic rat serum, ranged from 0.1 to 4.0 ng/ml with a mean of 1.76 ng/ml.