This Vitamin D3 (VD3) BioAssay™ ELISA Kit is a competitive ELISA for the in vitro quantitative determination of VD3 concentrations in serum, plasma and other biological fluids.||Sensitivity:|0.94ng/ml||Detection Range:|1.56-100ng/ml||Precision:|Intra-Assay %CV: <10%|Inter-Assay %CV: <10%||Specificity:|Recognizes VD3. No significant cross-reactivity or interference between VD3 and analogues was observed.||Test Principle:|The micro ELISA plate provided in this kit has been pre-coated with VD3. During the reaction, VD3 in the sample or standard competes with a fixed amount of VD3 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VD3. 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 VD3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.||KitComponents:|Micro ELISA Plate (Dismountable), 8 wells ×12 strips|Reference Standard, 2 vials|Concentrated Biotinylated Detection Ab (100×), 1x120ul|Concentrated HRP Conjugate (100×), 1x120ul|Reference Standard & Sample Diluent, 1x20ml|Biotinylated Detection Ab Diluent, 1x14ml|HRP Conjugate Diluent, 1x14ml|Concentrated Wash Buffer, (25×) 1x30ml|Substrate Reagent, 1x10ml|Stop Solution, 1x10ml ||Storage and Stability:|Unused kit is stable for 6 months after receipt.  Store Microtiter Strips, Standard, and Antibody-Biotin at 4°C if kit will be used within 30 days; otherwise store at -20°C until expiration date. Store the rest of the components at 4°C. Protect HRP Conjugate and Substrate Reagent from light. For maximum recovery, centrifuge the original vials after thawing and prior to removing the cap.||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.

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