|Hollis, Bruce - MED UNIV SC, CHARLESTON|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: December 3, 1998
Publication Date: N/A
Technical Abstract: The most abundant metabolite in plasma in the normal state or during vit D excess is 25-OH-D. C25 hydroxylation of vit D occurs in the liver microsomes and is the first step in the activation of vit D. The relatively high circulating concentrations of 25-OH-D makes this metabolite a useful clinical indicator of vit D status in states of nutritional vit D excess or deficiency, as well as during disease or treatment states such as enterohepatic disorders, anticonvulsant drug therapy, G.I. diseases or surgery, or liver and renal diseases. In 1993, Hollis et al. reported a RIA for 25-OH-D which used 125-I tracer and required a 1-step extraction with ACN. The method described here utilizes the same extraction protocol; however, assays were performed using a 96-wel te format and detection was achieved by chemiluminescence. Goat anti-25-OH-D 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. 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-OH-D conjugated to strepavidin HRP. Folowing a 2-h incubation, the wells were emptied, washed and treated with 150 ul of Supersignal**TM. Light emissions were detected using a Wallac 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, and the range of normal laboratory volunteers was 15-30 ng/ml.