Tumor necrosis factor (TNF) is a protein with a molecular weight of 17,000 produced by activated monocyte macrophages stimulated by endotoxin. Among them, TNF-α produced by macrophages is a cytokine that mediates cytotoxic effects by macrophages; TNF-β produced by lymphocytes. TNF has a wide range of biological activities in vivo: it can be used as an intermediate medium in the immune response, which has both anti-disease and pathogenic effects; it can cause hemorrhagic necrosis of tumors; it can stimulate fibroblast proliferation; -A, HLA-B, HLA-C antigen expression; can activate neutrophils and killer cells; can promote B cell proliferation and differentiation, increase IL-2 receptor expression. The liver is the main source of TNF and is also the main site of TNF clearance. At present, TNF-α is of great clinical significance and is an important mediator of liver damage caused by endotoxin. TNF-α can only exert toxic effects by specifically binding to the membrane-type receptor (mTNF-aR) on the surface of target cells. This binding process is regulated and restricted by the soluble TNF-α receptor (sTNF-aR) in serum. While sTNF-aR competes with mTNF-aR for binding to TNF-. . Clinically, TNF-α is mainly detected. Vet Autoclave,Best Dental Autoclave,Portable Dental Autoclave,Dental Sterilization Machine ZHEJIANG FOMOS MEDICAL TECHNOLOGY CO.,LTD. , https://www.ifomos.com
[Detection method] ELISA method
[Methodological principle] The anti-human TNF-α monoclonal antibody is coated on the microporous reaction plate, and the TNF-a in the test sample and the standard will be combined with the monoclonal antibody, the free component is washed away, and the biological substance is added at the same time. An anti-human TNF-α antibody and a horseradish peroxidase-labeled avidin. Biotin specifically binds to avidin, and the anti-human TNF antibody binds to human TNF-α bound to the monoclonal antibody to form an immune complex, and the free component is washed away. Adding a color developer, if there is TNF-α in the reaction well, HRP will make the colorless developer appear blue, and the stop solution turns yellow. The absorbance was measured at a wavelength of 450 nm, and the TNF concentration was proportional to the absorbance, and the concentration of TNF-α in the specimen was determined by plotting a standard curve.
[Preparation of specimen] 2ml of venous blood, not anticoagulation.
[Reagents]
1. Pre-coated microporous reaction plate
2. Concentrated enzyme conjugate
3. Enzyme conjugate dilution
4. Standard and specimen dilution
5. Concentrated biotinylated antibody
6. IFN standard crystal
7. Concentrated washing solution
8. Developers A, B
9. Stop solution
[Instrument] Microplate reader or fully automatic enzyme-linked immunosorbent detector.
[Detection step]
1. The sample to be tested, 100txl of different concentration standards, and 50tzl of biotinylated antibody working solution were added to the corresponding wells of the microporous reaction plate.
2. Shake well and incubate for 120 min at 20-25 °C.
3. The liquid in the well was blotted dry, washed thoroughly with washing solution 5 times, and dried.
4. In addition to the blank wells, the enzyme conjugate working solution 100u1 was added.
5. Shake well and incubate for 30 min at 20-25 °C.
6. The liquid in the well was blotted dry, washed thoroughly with washing solution 5 times, and dried.
7. Add 50 ul of each of the color developing agents A and B to each well, gently shake and mix, and color-proof for 10 min in a 37 C environment.
8. After adding 50 ul of the stop solution, gently shake and mix. The absorbance of each well was measured with a microplate reader (450 nm wavelength), and the TNF value was determined by comparison with a standard curve.
[Normal reference value] Each laboratory can establish a normal reference value depending on the detection method.
[Precautions]
1. When the kit is taken out of the refrigerated environment, it should be used in a room temperature environment for 30 minutes before use. The unused microporous strips are sealed with a ziplock bag.
2. When the enzyme plate is washed, each well needs to be filled with the washing liquid to prevent the free enzyme in the hole from being washed. The washing soaking time of 30 to 60 s should be set.
3. The drop bottle should be inverted several times before adding the reagent to mix the liquid. The bottle should be kept vertical when dropping to make the drop amount accurate.
4. All samples and waste should be disposed of as a source of infection. The stop solution is 2mol/L sulfuric acid and should be used safely.
5. A standard curve should be made for each batch of samples.
6. If the OD value of the specimen is outside the measurement range of the standard curve, it should be diluted and retested.
Detection of tumor necrosis factor by ELISA