Tumor Necrosis Factor alpha (TNF-alpha) BioAssay(TM) ELISA Kit (Human)

The Tumor necrosis factor alpha (TNF-alpha) BioAssay(TM) ELISA Kit (Human) is a sandwich ELISA for the quantitative determination of human tumor necrosis factor alpha (TNF-alpha) concentrations in serum, plasma, cell culture supernates, tissue homogenates, cell lysates. Sensitivity: 1.95pg/ml, Detection Range: 7.8-500pg/ml, Specificity: This assay has high sensitivity and excellent specificity for detection of human TNF-alpha. No significant cross-reactivity or interference between human TNF-alpha and analogues was observed. Precision: Intra-assay: CV%<8%, Inter-assay: CV%<10%, Test Principle: An antibody specific for TNF-alpha has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-alpha present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for TNF-alpha is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TNF-alpha bound in the initial step. The color development is stopped and the intensity of the color is measured, Kit Components: Assay plate, 1x96wells, Standard (Freeze dried), 2, Biotin-antibody (100X), 1x120ul, HRP-avidin (100X), 1x120ul, Biotin-antibody Diluent, 1x15ml, HRP-avidin Diluent, 1x15ml, Sample Diluent, 1x50ml, Wash Buffer (25X), 1x20ml, TMB Substrate, 1x10ml, Stop Solution, 1x10ml, Storage and Stability: Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Assay Summary: 1. Prepare reagents, standards and samples as instructed., 2. Add 100ul standard or sample to each well. Incubate for 1 hour at 37°C, 3. Aspirate and wash 3 times., 4. Add 100ul MAU/ALB (Biotin), 1X to each well. Incubate for 1 hour at 37°C, 5. Aspirate and wash 3 times., 6. Add 100ul Avidin (HRP), 1X to each well. Incubate for 1 hour at 37°C, 7. Aspirate and wash 5 times., 8. Add 90ul TMB substrate to each well Incubate for 20 minutes at 37°C. Protect from light, 9. Add 50ul Stop Solution to each well. Read at 450nm within 5 minutes.