The VB12 （Vitamin B12） BioAssay ELISA Kit is a quantitative competitive assay for the detection of Vitamin B12 in serum，plasma，tissue homogenates and other biological fluids.Range：0.781-50ng/mlSensitivity：<0.469ng/mlPrecision：Intra-assay CV： <8%Inter-assay CV： <10%Assay Principle：This ELISA kit employs the quantitative competitive enzyme-linked immunoassay technique，utilizing a microtiter plate provided in this kit has been pre-coated with an antibody specific to Vitamin B12. Standards and samples are added to the appropriate microtiter plate wells followed by a biotin-conjugated antibody specific to Vitamin B12. Streptavidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. After TMB substrate solution is added，only those wells that contain Vitamin B12，Biotin-conjugated antibody and enzyme-conjugated Streptavidin 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 Vitamin B12 in the sample is then determined by comparing the O.D. of the sample to the standard curve.Kit Components：*359604A： Microtiter Strips，1x96 wells （8x12 wells）.*359604B： Standard，2x1 vials359604C： Sample/Standard Dilution Buffer，1x20ml359604D： Antibody （Biotin） （Concentrated），1x60ul359604E： Antibody Dilution Buffer，1x10ml359604F： Streptavidin （HRP） （SABC），1x120ul359604G： SABC Dilution Buffer，1x10ml359604H： TMB Substrate，1x10ml359604J： Stop Solution，1x10ml359604K： Wash Buffer，25X，1x30mlStorage and Stability：Store unopened *359604A at 4ºC; store at -20ºC once opened. Store unopened *359604B at 4°C; once reconstituted，store at 4°C for up to 12 hours or at -20°C for up to 48 hours. Store other components at 4°C. Kit is stable for 6 months after receipt. For maximum recovery of product，centrifuge the original vials after thawing and prior to removing the cap.Assay Summary：1. Wash plate 2 times before adding standards，samples and control （zero） to wells!2. Add 50ul standard or sample to each well. Immediately add 50ul （Biotin） working solution to each well.3. Incubate for 45 minutes at 37°C4. Aspirate and wash 3 times.5. Add 100ul SABC working solution to each well. Incubate for 30 minutes at 37°C6. Aspirate and wash 5 times.7. Add 90ul TMB substrate. Incubate 10-20 minutes at 37°C8. Add 50ul Stop Solution. Read at 450nm immediately.9. Calculate results.
