The Human Vitamin D Binding Protein (DBP) ELISA Kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of DBP in human serum, plasma and other biological fluids. Detection Range: 0.156-10ng/ml Sensitivity: < 0.076ng/ml Precision: Intra-Assay: CV<10% Inter-Assay: CV<12% Test Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to DBP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to DBP. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain DBP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm 10nm. The concentration of DBP in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *028897A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use *028897B: Standard, 2x1vial 028897C: Standard Diluent, 1x20ml *028897D: Detection Reagent A, 1x120ul *028897E: Detection Reagent B, 1x120ul 028897F: Assay Diluent A, 1x12ml 028897G: Assay Diluent B, 1x12ml 028897H: TMB Substrate, 1x9ml 028897K: Stop Solution, 1x6ml 028897L: Wash Buffer, 30x, 1x20ml Storage and Stability: Store *028897A, *028897B, *028897D and *028897E at -20C. Store all the other components at 4C. 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. Materials Required But Not Supplied: 1. Microplate reader with 450 10nm filter. 2. Precision single or multi-channel pipettes and disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution Assay Procedure Summary: 1. Prepare all reagents, samples and standards. 2. Add 100ul standard or sample to each well. Incubate 2 hours at 37C. 3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37C. 4. Aspirate and wash 3 times. 5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37C. 6. Aspirate and wash 5 times. 7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37C. 8. Add 50ul Stop Solution. Read at 450nm immediately.
