This kit is for research use only
Detection range: 78 pg / ml-5000 pg / ml
Minimum detection limit: 19.5 pg / ml
Specificity: This kit can detect natural or recombinant human TF at the same time, and does not cross-react with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of TF content in human serum, plasma, cell culture supernatant or other related biological fluids.
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.
3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
Overview TF is a single-stranded transmembrane glycoprotein with a molecular weight of 47 kD and composed of 263 amino acid residues. Its gene is located on chromosomes 1p21 ～ 1p22 and consists of 6 exons and 5 introns. TF is named CD142 according to the cell surface antigen, belongs to the class II cytokine superfamily, and has the function of signal transduction. TF is composed of three domains: extracellular domain, transmembrane domain and cytoplasmic domain: (1) The extracellular domain is composed of 219 amino acid residues, including 2 disulfide bonds and 4 key amino acids binding FⅦ / FⅦa Residues, the latter is the key site to initiate the cascade of exogenous coagulation. (2) The transmembrane region is a hydrophobic structure composed of 23 amino acid residues, tightly bound to phospholipids, and its function is not yet clear. (3) The intracellular region is composed of 21 amino acid residues, among which there are three serine residues closely related to TF functional activity, which can be activated by phosphorylation by activated protein kinase C (PKC). TF, also known as factor Ⅲ, is a membrane receptor for the serine protease coagulation factor FⅦ / FⅦa. It binds to FⅦ to promote its conversion to FⅦa and forms a TF / Ⅶa complex. The TF / FVIIa complex further hydrolyzes inactive FXX into active FXXa, which combines with FVa and enzymatically cleaves prothrombin to thrombin on the surface of activated platelet phospholipid membrane to initiate the exogenous coagulation process. TF-expressing monocyte microparticles bind to activated platelets through their P-selectin glycoprotein ligand (PSGL-1) interacting with platelet P-selectin, and play an important role in the process of thrombosis.
TF is widely expressed in various tissue cells, such as endothelial cells, monocytes, macrophages and tumor cells. Under normal physiological conditions, TF is in an inactive state on the cell surface. Platelets and monocytes in the blood do not express TF, and the concentration of activated TF may be below 20 fmol / L. Under pathological conditions such as inflammation, tumor, and hypoxia, many active substances such as histamine, tumor necrosis factor alpha (TNF-α), and C-reactive protein (CRP) can induce vascular endothelial cells to express TF. Studies have shown that TF is abnormally increased on the surface of tumor cells such as pancreatic cancer, breast cancer, acute leukemia and glioma and vascular endothelial cells, and participates in the occurrence of tumor biological behavior and the formation of tumor microenvironment. Compared with non-epithelial malignant tumors, cancer cells derived from epithelial tissue express higher TF. Guan et al.'S study of 34 cases of glioma showed that the expression of TF at the protein and gene levels of tumor tissue increased significantly, and increased with the increase of tumor grade, but not expressed in normal brain tissue.
Experimental principle The microtiter plate is coated with purified antibody to make a solid-phase carrier, and the specimen or standard, biotinylated anti-TF antibody, and HRP-labeled avidin are sequentially added to the microwell coated with anti-TF antibody. After thorough washing, the color is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with TF in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Standard product (Standard): 2 bottles (lyophilized product).
3. Sample Diluent: 1 × 20ml / bottle.
4. Biotin-antibody Diluent: 1 × 10ml / bottle.
5. HRP-avidin Diluent: 1 × 10ml / bottle.
6. Biotin-antibody: 1 × 120μl / bottle (1: 100)
7. Horseradish peroxidase-labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100)
8. Substrate solution (TMB Substrate): 1 × 10ml / bottle.
9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4).
Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Collection and preservation of specimens such as distilled water and volumetric flasks
1. Serum: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000g for 20 minutes. The supernatant can be taken for detection, or the specimens should be stored at -20 ° C or -80 ° C, but repeated freezing should be avoided melt.
2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 1000g at 2-8 ° C for 15 minutes within 30 minutes after collection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing. melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000g for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Only when it is diluted to the range of the standard curve, the test result is accurate. Detailed records should be made during the dilution process. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".
Standard dilution principle: 2 bottles, dilute each bottle with sample diluent to 1ml before use, cover and let stand for more than 10 minutes, then repeatedly invert / scrub to help dissolve, its concentration is 5000 pg / ml, do After serial dilution, 5000 pg / ml, 2500 pg / ml, 1250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml, the sample dilution is directly used as the standard concentration 0 pg / ml, prepared within 15 minutes before use.
For the preparation of 2500 pg / ml standard: take 0.5ml (not less than 0.5ml) of the above standard at 5000 pg / ml and add it to the Eppendorf tube containing 0.5ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.
Dilution principle of biotinylated antibody:
Before use, dilute with biotin-labeled antibody diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). In actual preparation, 0.1-0.2ml should be prepared. For example, 10 μl of biotin-labeled antibody plus 990 μl of biotin-labeled antibody dilution is prepared, mixed gently, and prepared within one hour before use.
Dilution principle of horseradish peroxidase-labeled avidin:
Before use, dilute with horseradish peroxidase-labeled avidin diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). In actual preparation, 0.1-0.2ml should be prepared . For example, 10 μl horseradish peroxidase-labeled avidin plus 990 μl horseradish peroxidase-labeled avidin dilution is prepared, mix gently, and prepare within one hour before use.
Before starting the experiment, please configure all reagents in advance. When diluting the reagents or samples, they should be mixed evenly. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells, and add 100μl of standard or test sample to the remaining wells. Be careful not to have air bubbles. Add samples to the bottom of the wells of the microtiter plate. Try not to touch the well walls. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid and spin dry without washing. Add 100μl of biotinylated antibody working solution to each well (proportion of 1μl of biotinylated antibody plus 99μl of biotinylated antibody dilution), mix gently and prepare within one hour before use), 37 ℃, 60 minutes.
3. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 200μl / per well, spin dry.
4. Add 100 μl of horseradish peroxidase-labeled avidin working solution (with biotin-labeled antibody working solution) to each well at 37 ° C for 60 minutes.
5. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 200μl / per well, spin dry.
6. Add 90μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, the first 3-4 wells of the standard product can be seen by the naked eye with a clear blue gradient, but the rear 3-4 wells do not develop color. Obviously, it can be terminated).
7. Add 50μl of stop solution to each well in sequence to stop the reaction (at this time the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Note:
1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.
4. Store unused microplates or reagents at 2-8 ° C. Standards, biotin-labeled antibody working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. Do not reuse diluted standard, biotin-labeled antibody working solution, or horseradish peroxidase-labeled avidin working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Inject at least 0.3ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Calculate the standard concentration as the abscissa (logarithmic coordinate), the OD value is the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample ; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample .
Precautions
1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.
2. The washing process is very important. Insufficient washing can easily cause false positives.
3. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole.
5. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
6. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused.
7. Please keep the substrate away from light.
8. Do not replace the reagents in the kit with reagents from other manufacturers.
Camping Lounge Chairs ,Outdoor Folding Aluminum Chair,Foldable Lawn Chairs,Portable Camping Chair
NINGBO JENNY IMPORT & EXPORT CO.,LTD , https://www.jenny-china.com