Human 8-hydroxydeoxyguanosine (8-OHDG) enzyme-linked immunosorbent assay kit Instructions for use Read this manual carefully before use. The enzyme-linked immunosorbent kit is based on the principle of biotin double antibody sandwich technology to detect human 8-hydroxydeoxyguanosine (8-OHDG), which can only be used for research purposes and should not be used for medical diagnosis. Uses: For the determination of 8-hydroxydeoxyguanosine (8-OHDG) in human serum, plasma and related liquid samples. Working principle The kit uses biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human 8-hydroxydeoxyguanosine (8-OHDG) in the sample. Add 8-hydroxydeoxyguanosine (8-OHDG) to the well-labeled wells pre-coated with human 8-hydroxydeoxyguanosine (8-OHDG) monoclonal antibody, and incubate; after incubation, add biotin-labeled anti-8 -OHDG antibody, which binds to streptavidin-HRP to form an immune complex, which is then incubated and washed to remove unbound enzyme, then substrate A, B, blue, and in acid Converted to the final yellow. The color depth is positively correlated with the concentration of human 8-hydroxydeoxyguanosine (8-OHDG) in the sample. Kit composition 1 standard (128ng/ml) 0.5ml 7 developer A solution 6ml2 standard dilution 3ml 8 developer B solution 6ml3 enzyme label coating plate 12 holes × 8 strips 9 stop solution 6ml4 streptavidin -HRP 6ml 10 Instructions 1 part 5 30 times concentrated washing solution 20ml 11 sealing film 2 sheets 6 biotinylated anti- 8-OHDG antibody 1ml 12 sealed bag 1 required and not provided reagents and equipment 1. 37 ° C incubator. 2. Standard specification microplate reader. 3. Precision pipettes and disposable tips 4. Distilled water, 5. Disposable test tube 6. Absorbent paper notes 1. The kit removed from 2-8 ° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme label is not used up after the plate is opened, the slats should be stored in a sealed bag. 2. The sampler should be used for each step and the accuracy should be corrected frequently to avoid test errors. 3. Strictly follow the instructions of the manual, the test results must be determined by the microplate reader reading. 4. To avoid cross-contamination, avoid reusing the tips and closures in your hand. 5. Other reagents that are not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty. 6. Substrate B is sensitive to light and avoids prolonged exposure to light. Washing method: Manually washing the plate: pry off the liquid in the microplate; pour a few layers of absorbent paper on the test bench, and take a few times with the microplate; and at least 0.35 ml of the diluted washing solution is injected into the hole. Soak for 1-2 minutes. Repeat this process several times as needed. Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use. Specimen requirements 1. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity. 2. 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 freeze-thaw procedures should be avoided. Diluting the standard: (This kit provides one original standard, the user should follow the instructions to dilute in the small tube): 64ng/ml (No. 5 standard) 120μl of the original standard added to the standard of 120μl Product dilution 32ng/ml (Standard No. 4) 120μl Standard No. 5 Add 120μl standard dilution 16ng/ml (No. 3 standard) 120μl Standard No. 4 Add 120μl standard dilution 8ng/ml (Standard No. 2) 120 μl of Standard No. 3 Add 120 μl of Standard Diluent 4 ng/ml (No. 1 Standard) 120 μl of No. 2 Standard Add 120 μl of Standard Diluent 2. The number of slats required is determined by the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and the double hole can be used as much as possible. 3. Loading: 1) blank well: blank control well without sample, biotinylated anti-8-OHDG antibody, streptavidin-HRP, only coloring agent A&B and stop solution, the other steps are the same; 2 Standard well: 50μl of standard product, 50μl of streptomycin-HRP (the biotin antibody has been integrated in the standard, so it is not added); 3) The sample hole to be tested: 40μl of the sample is added, then anti- 8- 10 μl of OHDG antibody, 50 μl of streptavidin-HRP, covered with a sealing membrane, gently shaken and mixed, and incubated at 37 ° C for 60 minutes. 4. Solution: 30 times concentrated washing solution was diluted with distilled water 30 times 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. Color development: Add 50 μl of the developer A to each well, then add 50 μl of the developer B, gently shake and mix, and develop at 37 ° C for 10 minutes in the dark. 7. Termination: 50 μl of stop solution was added to each well to terminate the reaction (in this case, the blue color turned yellow). 8. Measurement: Zeroing was performed with a blank hole, and the absorbance (OD value) of each well was measured in order of 450 nm wavelength. The measurement should be carried out within 10 minutes after the addition of the stop solution. 9. Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. It can also be calculated using various application software. Summary of operating procedures: Prepare reagents, samples and standards, add prepared samples and standards, biotin-labeled secondary antibody and enzyme-labeled reagent, wash plate at 37 °C for 60 minutes for 5 minutes, add coloring solution A, B, 37 °C Color development 15 minutes into the stop solution within 10 minutes to read the OD value to calculate the detection range: 0.5 ng / ml → 100 ng / ml. Specifications: 96T / box storage: 2-8 ° C Validity: 6 months (2-8 ° C).
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