Instruction Manual of Mouse Inducible Nitric Oxide Synthase (iNOS) ELISA Kit This kit is for research use only Intended application Quantitative determination of iNOS in mouse serum, plasma, cell culture supernatant or other related liquids by ELISA. Experimental principle This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the iNOS level in the specimen. The microtiter plate was coated with purified antibody to make a solid-phase antibody. INOS, biotinylated anti-mouse iNOS antibody, and HRP-labeled avidin were added to the monoclonal antibody-coated microwells in turn. Substrate TMB color development. 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 iNOS 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. ELISA plate: one piece (96 wells) 2. Standard product (freeze-dried product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and left to stand for more than 10 minutes after being capped, then repeatedly inverted / rubbed to help dissolve, its concentration is 80 U / mL, after serial dilution, it is diluted to 80 U / mL, 40 U / mL, 20 U / mL, 10 U / mL, 5 U / mL, 2.5 U / mL, 1.25 U / mL As the highest standard concentration directly, the sample diluent is directly used as the standard concentration 0 U / mL, prepared within 15 minutes before use. For example, to prepare a 40 U / mL standard: take 0.5 ml of 80 U / mL of the above standard and add it to an Eppendorf tube containing 0.5 ml of sample diluent, mix well, and so on for the remaining concentrations. 3. Sample diluent: 1 × 20ml / bottle. 4. Test the diluent A: 1 × 10ml / bottle. 5. Test diluent B: 1 × 10ml / bottle. 6. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use. 7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use. The dilution method is the same as that of Test Solution A. 8. Substrate solution: 1 × 10ml / bottle. 9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water. 10. Stop solution: 1 × 10ml / bottle (2N H2SO4). Collection and preservation of specimens 1. Supernatant of cell culture: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing. 2. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing. 3. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 xg at 2-8 ° C for 15 minutes within 30 minutes after the specimen is collected, or store the specimen at -20 ° C, but avoid repeated freezing and thawing. Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested. Steps Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. 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 100ul of sample diluent to the blank well, and 100ul of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. 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 100ul of detection solution A working solution to each well (take 1ul of detection solution A plus 99ul of detection dilution A to prepare, mix gently and prepare within one hour before use), 37 ℃, 60 minutes. 3. After incubation for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per well, spin dry. 4. Add 100 μl of testing solution B working solution (same as testing A working solution) to each well at 37 ℃ 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, 350ul / per well, spin dry. 6. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient, and the latter 3-4 wells have no obvious gradient) , You can terminate). 7. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, 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. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring. 2. In order to prevent the sample from evaporating, the reaction plate is placed in a closed box covered with a damp cloth during the test, and the enzyme plate is covered with a cover or film. 3. Store unused microplates or reagents at 2-8 ° C. Standard products, working solution A, and working solution B should be configured and used according to the required amount. Do not reuse the diluted standard, test solution A working solution or test solution B working solution. 4. It is recommended to set a double-hole test 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 liquid into the hole, soak for 1-2 minutes, and repeat this process several times as needed. Specificity The kit can detect recombinant or natural mouse iNOS at the same time, and has no cross-reactivity with other related proteins. Calculation Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as 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 to obtain the actual concentration of the sample. Precautions 1. The washing process is very important, inadequate washing is easy to cause false positives. 2. 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. 3. Please make a standard curve at the same time of each measurement, it is best to make a double hole. 4. 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. 5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused. 6. Please keep the substrate away from light. examination range: 1.25 U / mL -80 U / mL Explanation 1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently). 2. Validity: 6 months 3. The concentrated washing liquid will have salt precipitation, and it can be heated and dissolved in the water bath when diluted. 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. 5. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions. 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Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
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