Vitamin D ELISA
Requiring no external sample preparation and no organic solvents, the Calbiotech 25-OH Vitamin D ELISA is a sensitive, robust, user friendly and automation-ready assay. Total assay time is 2.5-hours, with all incubations performed at room temperature. This test is based on the principle of competitive binding. Anti-Vitamin D antibody-coated wells are incubated with Vitamin D standards, controls, samples, and vitamin D-Biotin conjugate at room temperature for 90 minutes. Following a wash step, bound Vitamin D-Biotin is detected with Streptavidin-HRP. Streptavidin-HRP conjugate immunologically bound to the well progressively decreases as the concentration of Vitamin D in the specimen increases. Unbound SA-HRP conjugate is then removed and the wells are washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 30 minutes, resulting in the development of blue colour. The colour development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm. In an independent study, the Calbiotech 25(OH) Vitamin D ELISA showed excellent correlation to LC/MS/MS (R2=0.949; N=20), and performed better than a highly referenced, commercially available ELISA (R2=0.8314; N=20).
