Premium
A SENSITIVE AND SELECTIVE RADIOIMMUNOASSAY FOR SERUM 24, 25‐DIHYDROXYCHOLECALCIFEROL IN MAN
Author(s) -
HUMMER L.,
CHRISTIANSEN C.
Publication year - 1984
Publication title -
clinical endocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.055
H-Index - 147
eISSN - 1365-2265
pISSN - 0300-0664
DOI - 10.1111/j.1365-2265.1984.tb00138.x
Subject(s) - radioimmunoassay , sephadex , chemistry , chromatography , conjugate , high performance liquid chromatography , dichloromethane , antiserum , detection limit , competitive binding , endocrinology , medicine , antibody , biochemistry , receptor , biology , solvent , enzyme , mathematical analysis , mathematics , immunology
SUMMARY We have developed a sensitive and selective radioimmunoassay for 24, 25‐dihydroxycholecalciferol (24,25(OH) 2 D 3 ). Antisera with a high titre and affinity to 24, 25(OH) 2 D 3 were raised in rabbits immunized with a protein conjugate of the 3‐hemisuccinate derivative of 24, 25(OH) 2 D 3 . 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) 2 D 3 in the ‘24, 25(OH) 2 D complex’ isolated by HPLC and containing the comigrating metabolites: 24, 25(OH) 2 D 3 , 24, 25(OH) 2 D 2 , 25(OH)D 3 ‐26, 23 lactone and 25, 26(OH) 2 D 2 . 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) 2 D 3 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) 2 D, 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.