Scintillation proximity assay for calcitriol in serum without high pressure liquid chromatography.
A rapid isolation step for 1,25-dihydroxyvitamin D3 without high pressure liquid chromatography (HPLC) and a sensitive radioimmunoassay (RIA) have been developed. The time required for extraction and isolation with a combination of Extrelut-1-minicolumns and Sep-Pak silica cartridges from as little as 0.5 ml serum is only 2 h. The assay can be counted after 8 h of incubation. It is performed in the vial that collects the eluate, thus eliminating transfer losses and errors. No separation of bound and free hormone is necessary before beta-counting in the scintillation proximity assay. The detection limit of the assay is 2.7 ng/l. The intra-assay coefficients of variation are 7.3% and 5.2% for samples with calcitriol concentrations of 31 and 148 ng/l, respectively. The inter-assay coefficients of variation are 11.3%, 13.3% and 16.1% for low (16 ng/l), medium (30 ng/l) and high (148 ng/l) control pool samples, respectively. Normal values for calcitriol range from 32 to 80 ng/l. Elderly subjects, patients with reduced kidney function and pregnant women were also evaluated for their calcitriol levels. This assay correlates well with a RIA employing HPLC prepurification and charcoal separation of bound/free calcitriol (r = 0.94).
Wildermuth
S
S
University of Heidelberg, Germany.
Dittrich
K
K
Schmidt-Gayk
H
H
Zahn
I
I
O'Riordan
J L
JL
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002117:Calcitriol / Q000097:blood*; D002845:Chromatography / Q000379:methods*; D005260:Female; D006801:Humans; D007106:Immune Sera; D008297:Male; D008875:Middle Aged; D011247:Pregnancy; D011817:Rabbits; D012016:Reference Values; D012588:Scintillation Counting / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep
D007106:Immune Sera; D002117:Calcitriol