This Vitamin D (VD) BioAssay™ ELISA Kit is a competitive ELISA for the in vitro quantitative determination of VD concentrations in serum, plasma and other biological fluids.
Specificity:Recognizes VD. No significant cross-reactivity or interference between VD and analogues was observed.
Test Principle:The micro ELISA plate provided in this kit has been pre-coated with VD. During the reaction, VD in the sample or standard competes with a fixed amount of VD on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VD. 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 VD in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Storage and Stability:An unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 100ul standard or sample to each well. Incubate 90 minutes at 37°C
3. Aspirate and add 100ul prepared Biotin Working Solution. Incubate 1 hour 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.
