Tumor Necrosis Factor Alpha (TNFa) BioAssay(TM) ELISA Kit (Human) (High Sensitivity)

This Human Tumor Necrosis Factor alpha (TNFa) ELISA Kit (High Sensitivity) is a sandwich enzyme immunoassay for the in vitro quantitative measurement of TNFa in human serum, plasma, tissue homogenates, cell lysates, cell culture superantants and other biological fluids. Test Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to TNFa. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to TNFa. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain TNFa, biotin-conjugated antibody and enzyme-conjugated Avidin 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 TNFa in the sample is then determined by comparing the O.D. of the sample to the standard curve. Detection Range: 1.56-100pg/ml, Sensitivity: <0.55pg/ml, Precision: Intra-Assay: CV<10%, Inter-Assay: CV<12%, Kit Components: *153206A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use, *153206B: Standard, 2x1vial , 153206C: Standard Diluent, 1x20ml, *153206D: Detection Reagent A, 1x120ul , *153206E: Detection Reagent B, 1x120ul , 153206F: Assay Diluent A, 1x12ml, 153206G: Assay Diluent B, 1x12ml, 153206H: TMB Substrate, 1x9ml, 153206K: Stop Solution, 1x6ml, 153206L: Wash Buffer, 30x, 1x20ml , Storage and Stability: Store *153206A, *153206B, *153206D and *153206E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap. Materials Required But Not Supplied: 1. Microplate reader with 450 ± 10nm filter., 2. Precision single or multi-channel pipettes and disposable tips., 3. Eppendorf Tubes for diluting samples., 4. Deionized or distilled water., 5. Absorbent paper for blotting the microtiter plate., 6. Container for Wash Solution, Sample Preparation and Storage: Serum:, Use a serum separator and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles. Plasma: Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles. Note: Serum/plasma samples require about 5-fold dilution (for example: 40ul sample + 160ul PBS). Samples should be diluted using 0.01M PBS (pH 7.0-7.2). , Tissue Homogenates:, The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weighed before homogenization. The tissues were minced to small pieces and homogenized in 5-10ml of PBS with a glass homogenizer on ice (micro tissue grinder works too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifuged for 5 minutes at 2500×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower. Cell Lysates: Cells must be lysed before assaying according to the following directions. , 1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). , 2. Wash cells three times in cold PBS. , 3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4X (or freeze cells at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) , 4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris. Cell Culture Supernatants and Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Assay Procedure Summary: 1. Prepare all reagents, samples and standards., 2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C., 3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C., 4. Aspirate and wash 3 times., 5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C., 6. Aspirate and wash 5 times., 7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37°C., 8. Add 50ul Stop Solution. Read at 450nm immediately.