Mouse tumor necrosis factor alpha (TNF-α) elisa kit instruction manual

Mouse tumor necrosis factor alpha (TNF-α) elisa kit Instructions for use Elisa kit Specifications: 48 well configuration / 96 well configuration standard dilution: 1.5ml × 1 bottle of enzyme standard reagent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) [mouse tumor necrosis factor alpha (TNF-α) elisa kit] This reagent is only for research use calculation: the concentration of the standard is the abscissa, the OD value is the ordinate, and the coordinate paper Draw a standard curve, find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or calculate the linear regression equation of the standard curve with the concentration of the standard and the OD value, and substitute the OD value of the sample. Equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample. Kit composition: sealing film: 2 pieces (48) / 2 pieces (96) Instructions: 1 part sealed bag: 1 standard: 2700ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C preservation enzyme Standard package: 1×48 1×96 2-8°C Preservation of sample dilution: 3ml×1 bottle 6 ml×1 bottle 2-8°C Preservation of developer A: 3ml×1 bottle 6 ml×1 bottle 2 Store chromogen B solution at -8 °C: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution: 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C Preservation concentrated washing solution: (20ml × 20 times) ×1 bottle (20ml×30 times)×1 bottle 2-8°C preservation experiment principle: The kit uses double antibody sandwich method to measure the level of tumor necrosis factor α (TNF-α) in the sample. The microporous plate was coated with purified mouse tumor necrosis factor alpha (TNF-α) antibody to prepare a solid phase antibody, and tumor necrosis factor alpha (TNF-α) was sequentially added to the microcapsule of the coated monoclonal antibody, and then HRP was added. The labeled tumor necrosis factor alpha (TNF-α) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with tumor necrosis factor alpha (TNF-α) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of mouse tumor necrosis factor alpha (TNF-α) in the sample was calculated by a standard curve. OBJECTIVE: This kit is used to determine the content of tumor necrosis factor alpha (TNF-α) in serum, plasma and related fluid samples. Service commitment: ? Delivery period: payment to delivery. Free technical advice and guidance during working hours? Please call to provide customers with sample testing services to maximize the effectiveness of the experimental results (free proxy). Storage conditions and expiration date: kit storage: 2-8 ° C | Validity: 6 months [mouse tumor necrosis factor alpha (TNF-α) elisa kit] sample processing and requirements: 1. Serum: room temperature blood natural coagulation 10- Centrifuge for 20 minutes (2000-3000 rpm) for 20 minutes. The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again. 2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. 3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to. 4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use. 6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. Operation steps 1. Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme labeling plate, 100 μl of standard is added to the first and second holes, and then added in the first and second holes. 50 μl of the standard dilution, mix; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells. Then, 50 μl of each of the third and fourth wells is discarded, and 50 μl of each is added to the fifth and sixth wells, respectively, and 50 μl of the standard dilution is added to the fifth and sixth wells, respectively. After mixing, 50 μl from each of the fifth and sixth holes is added to the seventh and eighth holes, respectively, and then 50 μl of the standard dilution is added to the seventh and eighth holes, respectively, and mixed from the seventh. 50 μl of the eighth well was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 1800 ng/L, 1200 ng/L, 600 ng/L, 300 ng/L, 150 ng/L, respectively). 2. Loading: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix. 3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes. 4. Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used. 5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry. 6. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color: first add coloring agent A50μl to each well, then add coloring agent B50μl, gently shake and mix, avoid light at 37 °C 15 minutes. 10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow). 11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution. [Mouse tumor necrosis factor alpha (TNF-α) elisa kit] Note: 1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip should be stored in a sealed bag. 2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing does not affect the result. 3. The sampler should be used for each step and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun. 4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5). 5. The sealing film is intended for single use only to avoid cross-contamination. 6. Keep the substrate away from light. 7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading. 8. All samples, washings and various wastes should be treated as infectious materials. 9. The different batch components of this reagent must not be mixed. 10. In the case of an English manual, the English manual shall prevail. Kit performance: 1. Sample linear regression and expected concentration correlation coefficient R value of 0.95 or more. 2. Within and within the batch should be less than 9% and 11% detection range: 0.2IU/L - 6IU/L

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