8-hydroxydeoxyguanosine (8-OHdG) enzyme-linked immunosorbent assay in rats - Master's thesis - Dissertation

Rat 8-hydroxydeoxyguanosine (8-OHdG) Enzyme-Linked Immunoassay Kit Instructions for Use This kit is for research use only. Detection range: Purpose of use: This kit is used to determine the content of 8-hydroxydeoxyguanosine (8-OHdG) in rat serum, plasma and related liquid samples. Experimental principle This kit uses a double antibody sandwich method to determine the level of 8-hydroxydeoxyguanosine (8-OHdG) in rats. The purified rat 8 hydroxydeoxyguanosine (8-OHdG) antibody was coated with a microplate to prepare a solid phase antibody, and 8 hydroxydeoxyguanosine (8-OHdG) was sequentially added to the microcapsule of the coated monoclonal antibody. Binding to an HRP-labeled 8-hydroxydeoxyguanosine (8-OHdG) antibody forms 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 8-hydroxydeoxyguanosine (8-OHdG) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat 8-hydroxydeoxyguanosine (8-OHdG) in the sample was calculated from a standard curve. Kit composition specimen requirements 1. 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. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity. Procedure 1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart. 2. Adding samples: 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), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, 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. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes. Solution: 30 times concentrated washing solution is diluted with distilled water 30 times and then washed. Carefully remove the sealing film, discard the liquid, dry it, fill each well with washing liquid, let stand for 30 seconds, then discard it, repeat 5 Times, shoot dry. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells. Incubation: The operation is the same as 3. Washing: The operation is the same as 5. Color development: Add 50 μl of the developer to each well, then add 50 μl of the developer B, gently shake and mix, and color at 37 ° C for 15 minutes. 10. Stop: add 50 μl of stop solution to each well to stop the reaction. Blue turns 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. Summary of operation procedure: Calculate the concentration of the standard object as the abscissa and the OD value as the ordinate. Draw a standard curve on the 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 Calculate the linear regression equation of the standard curve by using the concentration of the standard and the OD value. Substituting the OD value of the sample into the equation, calculating the sample concentration, and multiplying by the dilution factor, is the actual concentration of the sample. Note 1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats 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 will 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. Please keep the substrate away from light. 7. Strictly follow the operation of the manual, the test results must be based on the reading of the microplate reader. 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. If it is different from the English manual, the English manual shall prevail. Storage conditions and validity period 1. The kit is stored at: 2-8 °C. 2. Validity: 6 months