Sample Type：Serum，plasma and other biological fluidsSpecificity：GeneralIntended Use：BioAssay ELISA Kit for Vitamin D3 （VD3） in serum，plasma and other biological fluids.Detection Range：4.94-400ng/mlSensitivity：<1.85ng/mlPrecision：Intra-Assay CV： <10%Inter-Assay CV： <12%Test Principle：This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for VD3 has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled VD3 and unlabeled VD3 （standards or samples） for limited binding sites on the pre-coated antibody. After incubation the unbound conjugate is washed off. Next，avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. The amount of bound HRP conjugate is inversely proportional to the concentration of VD3 in the sample. After addition of the substrate solution，the intensity of color developed is inversely proportional to the concentration of VD3 in the sample.Kit Components：*366700A： Microtiter Plate，96 wells，Pre-coated，ready to use.*366700B： Standard，2x1vial 366700C： Standard Diluent，1x20ml*366700D： Detection Reagent A，1x1vial *366700E： Detection Reagent B，1x120ul 366700F： Assay Diluent A，1x12ml366700G： Assay Diluent B，1x12ml366700H： TMB Substrate，1x9ml366700K： Stop Solution，1x6ml366700L： Wash Buffer，30X，1x20ml366700M： Reagent Diluent，1x300mlStorage and Stability：Store *366700A，*366700B，*366700D and *366700E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. 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，then add 50ul prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37°C.3. Aspirate and wash 3 times.4. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.5. Aspirate and wash 5 times.6. Add 90ul Substrate Solution. Incubate 15-25 minutes at 37°C.7. Add 50ul Stop Solution. Read absorbance at 450nm immediately.
