Tartrate-Resistant Acid Phosphatase 5b （TRACP-5b） BioAssay ELISA Kit is a quantitative sandwich assay for the detection of TRACP-5b in mouse serum，plasma，tissue homogenates and other biological fluids.Detection Range：0.781-50ng/mlSensitivity：<0.469ng/mlPrecision： Intra-Assay CV： <8%Inter-Assay CV： <10%Kit Components：*359452A： Microtiter Strips，1x96 wells （8x12 wells）.*359452B： Standard，2x1 vial359452C： Sample/Standard Dilution Buffer，1x20ml359452D： Antibody （Biotin） （Concentrated），1x120ul359452E： Antibody Dilution Buffer，1x10ml359452F： Streptavidin （HRP） （SABC），1x120ul359452G： SABC Dilution Buffer，1x10ml359452H： TMB Substrate，1x10ml359452J： Stop Solution，1x10ml359452K： Wash Buffer，25X，1x30mlStorage and Stability：Store unopened *359452A at 4ºC; store at -20ºC once opened. Store unopened *359452B 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 Principle：This ELISA kit employs the sandwich enzyme-linked immunoassay technique，utilizing a microtiter plate pre-coated with an antibody specific to TRACP-5b. Standards and samples are added to the appropriate wells，then incubated. Aspirate and wash the plate. The Antibody （Biotin） is added to all the wells. The plate is incubated and then washed. Streptavidin （HRP） is added to each microplate well and incubated then washed. Then the TMB substrate solution is added and incubated. After the TMB substrate solution is added，only those wells that contain TRACP-5b，the antibody （Biotin） and Streptavidin （HRP） 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 TRACP-5b in the sample is then determined by comparing the O.D. of the sample to the standard curve.Assay Summary：1. Wash plate 2 times before adding standards and samples to wells!2. Add 100ul standard or sample to each well and incubate for 90 minutes at 37°C. Aspirate and wash 2 times.3. Add 100ul Antibody （Biotin） working solution to each well and incubate for 60 minutes at 37°C. 4. 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 15-30 minutes at 37°C8. Add 50ul Stop Solution. Read at 450nm immediately.9. Calculate results.
