25-OH Vitamin D ELISA 试剂盒

Before running the test, prepare the following:
1. Standards and Reagents: Standards are serum-based solutions and stable when stored at -2-8°C, protected from light, until the expiration date on the label. Equilibrate the needed volume of standards and reagents to room temperature before use.
2. 51X Biotin conjugate: Immediately before use, prepare 1X working solution at 1:51 with assay diluent (e.g. Add 0.1ml of the 50x Vitamin D-Biotin conjugate concentrate to 5ml of assay diluent). Remaining Assay Diluent must be stored at 2-8°C in dark and tightly capped.
3. Prepare 1X Wash Buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-24 °C).

• 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.
• During the incubation, a fixed amount of biotin-labeled vitamin D competes with the endogenous Vitamin D in the sample, standard, or quality control serum for a fixed number of binding sites on the anti Vitamin D antibody.
• 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 color.
• The color development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm.
• A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The color intensity will be inversely proportional the amount of 25(OH)D in the sample.

Results are expressed in ng/mL. Note: To convert to nmol/L, multiply results by 2.5. Example: 10 ng/mL = 25 nmol/L.