The Pro-endothelin 1 （Pro-ET1） BioAssay ELISA Kit （Human） is a quantitative sandwich assay for the detection of Pro-ET1in human serum，plasma，tissue homogenates and other biological fluids.Detection Range：0.871-50pg/mlSensitivity：<0.469pg/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 Pro-ET1. Standards and samples are added to the appropriate microtiter plate wells followed by a biotin-conjugated antibody specific to Pro-ET1. 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 Pro-ET1，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 Pro-ET1 in the sample is then determined by comparing the O.D. of the sample to the standard curve.Kit Components：*369676A： Microtiter Strips，8x12 wells.*369676B： Standard，2 vials369676C： Sample/Standard Dilution Buffer，1x20ml369676D： Antibody-Biotin （Concentrated），1x120ul369676E： Antibody Dilution Buffer，1x10ml369676F： Streptavidin-HRP Conjugate （SABC），1x120ul369676G： SABC Dilution Buffer，1x10ml369676H： TMB Substrate，1x10ml369676J： Stop Solution，1x10ml369676K： Wash Buffer （25X），1x30mlStorage and Stability：Store unopened *369676A at 4ºC; store at -20ºC once opened. Store unopened *369676B 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.Precautions for Use：1. To inspect the validity of experiment operation and the appropriateness of sample dilution，conducting a pilot experiment using standards and a small number of samples is recommended.2. Keep plate dry after opening and before using.3. Before using the kit，spin tubes and bring down all components to the bottom of tubes.4. Store TMB reagents in the dark.5. Washing process is very important. False positive results can be caused by not doing complete washes. 6. Duplicate wells are recommended for both standards and sample testing.7. Do not let plate go dry during the assay because this will inactivate active components.8. Do not reuse tips and tubes to avoid cross contamination.9. Avoid using the reagents from different batches together.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°C.3. Aspirate and wash plate 2 times.4. 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 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.
