Instruction Manual for Rat S100B Protein (S100B) ELISA Kit This kit is for research use only Detection range: 312 pg / ml-20000 pg / ml Minimum detection limit: 78 pg / ml Specificity: This kit can detect natural or recombinant rat S100B at the same time, and does not cross-react with other related proteins. Validity: 6 months Intended application: ELISA method for quantitative determination of S100B in rat serum, plasma, cell culture supernatant or other related biological fluids. Explanation 1 . Store the kit: -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. Experimental principle Coat the microplate with purified antibody to make a solid-phase carrier. Add specimens or standards, biotinylated anti-S100B antibody, and HRP-labeled avidin to the microwells coated with anti-S100B antibody in sequence. After washing, it was 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 S100B in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration. Kit composition and reagent preparation Reagents and equipment needed but not provided 6. Distilled water, volumetric flask, etc. Collection and preservation of specimens 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 the concentration is finally calculated, it is diluted by "N" times, and the concentration of the specimen should be multiplied by "N". Standard dilution principle: 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and then left to stand for more than 10 minutes after capping, and then reversed / rubbed repeatedly to help dissolve, its concentration is 20000 pg / ml, do After serial dilution, dilute 20,000 pg / ml, 10000 pg / ml, 5000 pg / ml, 2500 pg / ml, 1250 pg / ml, 625 pg / ml, 312 pg / ml, and the sample diluent is directly used as the standard concentration 0 pg / ml, prepared within 15 minutes before use. For example, to prepare 10000 pg / ml standard: take 0.5ml (not less than 0.5ml) of the above 20000 pg / ml standard into 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, prepare 10μl biotin-labeled antibody plus 990μl biotin-labeled antibody dilution, mix gently, and prepare within one hour before use. Dilution principle of horseradish peroxidase-labeled avidin: Dilute with horseradish peroxidase-labeled avidin diluent before use. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). 0.1-0.2ml should be prepared during actual preparation . For example, prepare 10μl horseradish peroxidase-labeled avidin plus 990μl horseradish peroxidase-labeled avidin dilution, mix gently, and prepare within one hour before use. 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. To ensure the validity of the experimental results, please use a new standard solution for each experiment. 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 first during measurement. 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. 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. 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.
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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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