A SENSITIVE AND SELECTIVE RADIOIMMUNOASSAY FOR SERUM 24, 25‐DIHYDROXYCHOLECALCIFEROL IN MAN

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Abstract
SUMMARY: 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.