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

Abstract

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.