Description
| Sample | Serum, plasma, cell culture supernates |
| Intended use | This Sandwich kit is for the accurate quantitative detection of Human Tumor Necrosis Factor alpha (also known as TNFA) in serum, plasma, cell culture supernatants, Ascites, tissue homogenates or other biological fluids. |
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Test principle |
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Human TNFA antibody. TNFA present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Human TNFA Antibody is added and binds to TNFA in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated TNFA antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Human TNFA. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm. |
| Specimen Collection | Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes. Collect the supernatant without sediment. Plasma Collect plasma using EDTA or heparin as an anticoagulant. After mix 10-20 minutes, centrifuge samples for 20 minutes at 2000-3000 RPM. Collect the supernatant without sediment.
Cell culture supernatant Collect by sterile tubes. When detecting secrete components, centrifuge at 2000-3000 RPM for 20 minutes. Collect the supernatants. When detecting the components in the cell, use PBS (pH 7.2-7.4) to dilute cell suspension, the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for 20 minutes. Collect the supernatant without sediment. Tissue Rinse tissues in ice-cold PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (tissue weight (g): PBS (mL) volume=1:9) with a glass homogenizer on ice. To further break down the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 15 minutes at 12,000 RPM at 4°C to get the supernatant. Avoid freeze/thaw cycles. Urine/Ascites/Cerebrospinal fluid Collect by sterile tube. Centrifuge at 2000-3000 RPM for 20 minutes. Collect the supernatant without sediment. |
| Storage/Stability | -20°C/1 year; 4°C/6 months |
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