The Tumor Necrosis Factor alpha （TNF-alpha） BioAssay ELISA Kit （Canine） is a quantitative sandwich assay for the detection of TNF-alpha in canine serum，plasma，tissue homogenates and other biological fluids.Sensitivity：<9.375pg/mlDetection Range：15.625-1000pg/mlPrecision： Intra-Assay CV： <8%Inter-Assay CV： <10%Assay Principle：The microtiter plate provided in this kit has been pre-coated with an antibody specific to TNF-alpha. Standards and samples are added to the appropriate microtiter plate wells followed by a biotin-conjugated antibody specific to TNF-alpha. 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 TNF-alpha，biotin-conjugated antibody and enzyme-conjugated streptavidin complex 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 TNF-alpha in the sample is then determined by comparing the O.D. of the sample to the standard curve.Kit Components：*359341A： Microtiter Strips，8x12 wells.*359341B： Standard，2 vials359341C： Sample/Standard Dilution Buffer，1x20ml359341D： Antibody-Biotin （Concentrated），1x120ul359341E： Antibody Dilution Buffer，1x10ml359341F： Streptavidin-HRP Conjugate （SABC），1x120ul359341G： SABC Dilution Buffer，1x10ml359341H： TMB Substrate，1x10ml359341J： Stop Solution，1x10ml359341K： Wash Buffer （25X），1x30mlStorage and Stability：Store unopened 359341 at 4ºC; store at -20ºC once opened. Store unopened *359341B 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 100ul standard or sample to each well and incubate for 90 minutes at 37°C3. Add 100ul Antibody-Biotin working solution to each well and incubate for 60 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 the plate 5 times.7. Add 90ul TMB substrate. Incubate the plate for 15-30 minutes at 37°C8. Add 50ul Stop Solution. Read at 450nm immediately.9. Calculate results.
