|Hollis, Bruce - MED UNIV SC, CHARLESTON|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: December 1, 1998
Publication Date: N/A
Technical Abstract: The most abundant metabolite in plasma in the normal state or during vit D excess is 25-hydroxyvitamin D (25-OHD). C25 hydroxylation of vit D occurs in the liver microsomes. The relatively high circulating concentrations of 25-OHD makes this metabolite a useful clinical indicator of vit D status in states of nutritional vit D excess or deficiency. As the clinical demand for circulating 25-OHD analysis increased, it was clear that simpler, rapid, and valid assay procedures would be required. In 1993, Hollis et al. reported an RIA for 25-OHD which used 125-I tracer and required a 1-step extraction with acetonitrile (ACN). The method described here utilizes the same extraction protocol; however, assays were performed using a 96-well plate format and detection was achieved by chemiluminescence. Goat anti-25-OHD antisera was immobilized by adding diluted antibody to wells pretreated with rabbit anti-goat IgG. The plates were washed with PBS and stored at -70 C. Prior to use, the wells were blocked using Superblock**TM. Samples were prepared for assay by adding 50 ul of standards or unknowns to 500 ul ACN. The samples were centrifuged and 30 ul of supernatant was removed and added to 600 ul of assay buffer. Two 250 ul aliquots of the diluted sample were placed into individual wells. After 2 h, the wells were emptied, washed and treated with 250 ul of biotinylated 25-OHD conjugated to strepavidin HRP. Following a 2-h incubation, the wells were emptied, washed and treated with 150 ul of Supersignal**TM. Light emissions were detected using a Wallac Victor**TM 1420 Multilabel Counter. The assay range was 2.5-100 ng/ml. Samples from patients of marginal vit D status ranged from 6-12 ng/ml; the range of normal laboratory volunteers was 15-30 ng/ml.