The 25-Hydroxy Vitamin D3 (25-HVD3) BioAssay™ ELISA Kit is a competitive ELISA for the in vitro quantitative determination of 25-HVD3 concentrations in serum, plasma and other biological fluids.
Specificity:Recognizes 25-HVD3. No significant cross-reactivity or interference between 25-HVD3 and analogues was observed.
Test Principle:The micro ELISA plate provided in this kit has been pre-coated with 25-HVD3. During the reaction, 25-HVD3 in the sample or standard competes with a fixed amount of 25-HVD3 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to 25-HVD3. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm. The concentration of 25-HVD3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Storage and Stability:Store *529892A, *529892B, *529892D and *529892E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 50ul standard or sample to each well
3. Immediately add 50ul prepared Biotin Working Solution. Incubate 45 minutes at 37°C
4. Aspirate and wash 3 times
5. Add 100ul prepared HRP Detection Working Solution. Incubate 30 minutes at 37°C
6. Aspirate and wash 5 times
7. Add 90ul Substrate. Incubate 15 minutes at 37°C
8. Add 50ul Stop Solution. Read at 450nm immediately.
