25-Hydroxy Vitamin D
25-OH Vitamin D EIA Kit is designed for the quantification of total 25-OH Vitamin D in human serum and plasma.
This innovative new assay uses enzyme immunoassay technology (EIA) to quantify total 25-OH vitamin D (D2+D3) from serum or plasma samples in about two hours. The test can be run on both manual and automated microplate readers. Test accuracy is assured by a fully automated extraction step that removes proteins in the sample that may interfere with test performance. The method is highly precise with Intra-assay %CV of ≤15% for samples ≥15 ng/ml. The test correlates well with the current methods. When tested with samples ranging from 11.9 ng/ml – 131.5 ng/ml the correlation coefficient was r2 = 0.930 with a slope of 0.941 and a y-intercept of 1.4 ng/ml. The assay is directly traceable to the industry standard NIST SRM 972 and has demonstrated excellent performance with the DEQAS proficiency program.
The assay is based on the competition between a 25-OH vitamin D conjugate coated on a microplate and the 25-OH Vitamin D content of a sample. Vitamin D is extracted (EX Reagent) from your samples and then mixed, in the coated microplate well breakaway plates, with reagent R1 which contains an antibody for 25-OH Vitamin D. After a first incubation, the microplate is washed and reagent R2 (HRP-labeled secondary antibody) is added. Following a second incubation and a washing step, reagent R3 (TMB substrate) is added. After a final incubation step, the reaction is stopped by adding the STOP solution and the colorimetric signal of the microplate is measured at 450 nm (primary wavelength) to which the background signal can be subtracted by using secondary wavelengths (620 to 650 nm). The 25-OH Vitamin D concentration of a patient sample is inversely proportional to the measured absorbance at 450 nm. The whole assay procedure takes about two hours.
- Holick, MF. Vitamin D Status: Measurement, Interpretation andClinical Application. Ann Epidemiol. 2009, 19(2): 73–78.
- National Osteoporosis Foundation. Prevention – Vitamin D. http://www.nof.org/aboutosteoporosis/prevention/vitamind