A radioimmunoassay for 1,25-dihydroxycholecalciferol.

We describe a radioimmunoassay for 1,25-dihydroxycholecalciferol in human serum. We raised antisera in rabbits to 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine serum albumin, and obtained sensitive, high-titer antibodies. These antibodies had a high affinity for 1,25-dihydroxycholecalciferol and cross reacted mainly with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Addition of 1 mL of normal rabbit serum per liter reduced this interference to 5 and 4%, respectively. However, these interfering steroids are present in large excess, so extensive purification of 1,25-dihydroxycholecalciferol from serum is necessary. The steroid was extracted with ethyl acetate/cyclohexane, purified on Sephadex LH-20, and then chromatographed on a column of silicic acid. The radioimmunoassay is sensitive to 5 pg/tube (3 ng/L of serum). The between-assay CV was 14%. The mean concentration of 1,25-dihydroxycholecalciferol in the serum of 54 healthy adults was 38 (SD 12) ng/L, with no sex-related difference. The assay was further validated by the finding of low or undetectable concentrations in patients with chronic renal failure and of increased concentrations in the serum of patients with primary hyperparathyroidism. In comparison with previously described methods, the major advantage of the present assay is the use of stable gamma-globulins, which are available in large amounts, as binding protein.

[1]  D. Bikle.  The vitamin D endocrine system. , 1982, Advances in internal medicine.

[2]  G. Hendy,et al.  MEASUREMENT OF 1,25‐DIHYDROXYCHOLECALCIFEROL IN MAN BY RADIOIMMUNOASSAY , 1979, Clinical endocrinology.

[3]  J. I. Pedersen,et al.  Assay of 1,25-dihydroxy vitamin D3 by isotope dilution--mass fragmentography. , 1979, Clinical chemistry.

[4]  H. DeLuca,et al.  A bioassay capable of measuring 1 picogram of 1,25-dihydroxyvitamin D3. , 1978, The Journal of clinical endocrinology and metabolism.

[5]  J. Lemann,et al.  The simultaneous measurement of vitamin D metabolites in plasma: studies in healthy adults and in patients with calcium nephrolithiasis. , 1978, The Journal of laboratory and clinical medicine.

[6]  T. Spelsberg,et al.  Isolation and quantitation of endogenous vitamin D and its physiologically important metabolites in human plasma by high pressure liquid chromatography. , 1977, Journal of steroid biochemistry.

[7]  H. DeLuca,et al.  1,25-Dihydroxyvitamin D in biological fluids: a simplified and sensitive assay. , 1976, Science.

[8]  H. DeLuca,et al.  A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma. , 1976, Archives of biochemistry and biophysics.

[9]  J. Haddad,et al.  25-Hydroxyvitamin D transport in human plasma. Isolation and partial characterization of calcifidiol-binding protein. , 1976, The Journal of biological chemistry.

[10]  M. Haussler,et al.  Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3. , 1976, The Journal of clinical investigation.

[11]  P. D. de Moor,et al.  The purification and characterisation of the human-serum binding protein for the 25-hydroxycholecalciferol (transcalciferin). Identity with group-specific component. , 1976, European journal of biochemistry.

[12]  I. Holmberg,et al.  A novel specific assay of 25-hydroxy vitamin D. , 1976, Clinica chimica acta; international journal of clinical chemistry.

[13]  P. D. de Moor,et al.  Measurement of 25-hydroxyvitamin D3 in serum. , 1976, Clinical chemistry.

[14]  G. Just,et al.  A SELECTIVE, MILD CLEAVAGE OF TRICHLOROETHYL ESTERS, CARBAMATES, AND CARBONATES TO CARBOXYLIC ACIDS, AMINES, AND PHENOLS USING ZINC TETRAHYDROFURAN PH 4.2-7.2 BUFFER , 1976 .

[15]  M. Haussler,et al.  Filter assay for 1alpha, 25-dihydroxyvitamin D3. Utilization of the hormone's target tissue chromatin receptor. , 1974, Biochemistry.

[16]  E. Rizzardo,et al.  Convenient synthesis of crystalline 1α,25-dihydroxyvitamin D3 , 1974 .

[17]  H. DeLuca,et al.  The synthesis of 1α,25-dihydroxycholecalciferol -- A metabolically active form of vitamin D3 , 1972 .

[18]  H. DeLuca,et al.  Competitive binding assay for vitamin D and 25-OH vitamin D. , 1971, The Journal of clinical endocrinology and metabolism.

[19]  J. Haddad,et al.  Competitive protein-binding radioassay for 25-hydroxycholecalciferol. , 1971, The Journal of clinical endocrinology and metabolism.

[20]  B. Erlanger,et al.  Steroid-protein conjugates. II. Preparation and characterization of conjugates of bovine serum albumin with progesterone, deoxycorticosterone, and estrone. , 1959, The Journal of biological chemistry.

[21]  M. Haussler,et al.  Radioligand Receptor Assay for 25-Hydroxyvitamin D2/D3 and la,25-Dihydroxyvitamin D2/D3 APPLICATION TO HYPERVITAMINOSIS D MMu , 2022 .