A new, highly sensitive assay for 1,25-dihydroxyvitamin D not requiring high-performance liquid chromatography: application of monoclonal antibody against vitamin D receptor to radioreceptor assay.
A new, highly sensitive radioreceptor assay, which does not require high-performance liquid chromatography, has been developed for the determination of 1,25-dihydroxyvitamin D3 (1,25-(OH2)D3) in serum. The assay involves rapid extraction of serum, Sep Pak silica purification, and addition of 1,25-dihydroxyvitamin D3 receptor, radiolabeled 1,25-dihydroxyvitamin D3, bovine serum albumin, and monoclonal antibody to specifically precipitate the receptor. This method is sensitive to 0.3-0.6 pg/tube, with B50 occurring at 5.8 pg/tube. This sensitivity combined with overall recovery of 1,25-dihydroxyvitamin D3 (81.5 +/- 5.2%, n = 50, mean +/- SD) allows the measurement of serum 1,25-(OH)2D3 in duplicates with only 0.5 ml of serum. Intra- and interassay coefficient of variation were 9.5 and 14.6%, respectively. Dilution analysis, analytical recovery of added 1,25-dihydroxyvitamin D3, and comparison with a standard method using HPLC have been used to validate the assay. Serum 1,25-dihydroxyvitamin D3 level was for normal adults, 36.6 +/- 10.5 pg/ml (n = 14); in primary hyperparathyroidism, 98.9 +/- 19.9 pg/ml (n = 16); in chronic renal failure, 17.8 +/- 5.1 pg/ml (n = 12). This method allows large numbers of samples to be processed at once. Further, the method is rapid and provides an accurate assay using small amounts of serum.
Koyama
H
H
Second Department of Internal Medicine, Osaka City University Medical School, Japan.
Prahl
J M
JM
Uhland
A
A
Nanjo
M
M
Inaba
M
M
Nishizawa
Y
Y
Morii
H
H
Nishii
Y
Y
DeLuca
H F
HF
D000284:Administration, Oral; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D002117:Calcitriol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006887:Hydroxycholecalciferols / Q000008:administration & dosage; D006961:Hyperparathyroidism / Q000097:blood; D007422:Intestines; D008297:Male; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000276:immunology*; D013552:Swine
D000911:Antibodies, Monoclonal; D006887:Hydroxycholecalciferols; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D002117:Calcitriol; C008088:alfacalcidol