Chinese manual ________________________________________________________ Rat glutathione (GSH) ELISA kit detection principle The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules pre-coated with the glutathione (GSH) capture antigen of the rat were sequentially added with the specimen, the standard, and the HRP-labeled detection antibody, and incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with rat glutathione (GSH) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration. Sample Collection, Handling and Preservation Method 1. Serum: Use a tube containing no pyrogen and endotoxin to avoid any cell irritation during the operation. After collecting the blood, centrifuge and centrifuge for 10 minutes at 3000 rpm to quickly and carefully separate the serum and red blood cells. 2. Plasma: EDTA, citrate or heparin anticoagulation. The supernatant was taken by centrifugation at 3000 rpm for 30 minutes. 3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymer. 4. Tissue homogenization: The tissue is added to the appropriate amount of physiological saline and chopped. The supernatant was taken by centrifugation at 3000 rpm for 10 minutes. 5. Storage: If the sample is not detected in time after collection, please dispense it once, freeze it at -20 °C, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly. Self-contained microplate reader (450nm) high-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL 37 °C incubator operation notes The kit is stored at 2-8 ° C, before use Allow to equilibrate for 20 minutes at room temperature. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage. The pretreated sample does not need to be diluted, and 10 μL of the sample can be directly added. Incubation is carried out in strict accordance with the time indicated in the instructions, the amount of liquid added, and the order. Shake well all liquid components before use. Kit Composition Name 96-well configuration 48-well configuration Remarks Microporous microplates 12 wells × 8 strips 12 wells × 4 strips without standard (80 ng/ml) 0.5 mL 0.5 mL Diluted standard dilutions 6 mL 3 mL according to the instructions Instructions for dilution of the sample dilution 6mL 3mL According to the instructions for dilution detection antibody-HRP 6mL 3mL without 20 × wash buffer 20mL 20mL according to the instructions to dilute the substrate A 6mL 3mL without substrate B 6mL 3mL no stop solution 6mL 3mL no seal film 2 sheets, 2 sheets, no instructions, 1 part, no zipper bags, 1 piece, no note: Standards are diluted with standard dilutions to: 80, 40, 20, 10, 5, 0 ng/ml. Preparation of reagents 20× Dilution of Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20X wash buffer plus 19 parts of distilled water. Washing the plate by hand: Wash the liquid in the hole, fill each hole with the washing liquid, let stand for 1 min, then drain the liquid in the hole, pat dry on the absorbent paper, and wash the plate 5 times. Automatic washing machine: Inject 350 μL of washing solution into each well, soak for 1 min, and wash the plate 5 times. Procedure The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag. Set standard hole, sample hole and blank hole, blank hole is added; standard sample hole is added with different concentration of standard product 50μL; sample hole to be tested is first added 10μL of sample to be tested, plus sample dilution 40μL; 50 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to the wells and sample wells (no blank cells were added), and the reaction wells were sealed with a sealing plate and incubated for 60 min in a 37 ° C water bath or incubator. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine). 50 μL of each of the substrates A and B was added to all wells, and incubated at 37 ° C for 15 min in the dark. 50 μL of the stop solution was added to all wells, and the OD value of each well was measured at a wavelength of 450 nm within 15 min. The result is judged to draw a standard curve: in the Excel worksheet, the standard product concentration is used as the abscissa, the corresponding OD value is plotted as the ordinate, the standard linear regression curve is drawn, and the sample concentration values ​​are calculated according to the curve equation. Kit performance accuracy: standard product linear regression and expected concentration correlation coefficient R value, greater than or equal to 0.9900. Sensitivity: The minimum detection concentration is less than 1.0 ng/ml. Specificity: Does not cross-react with other soluble structural analogs. Repeatability: The coefficient of variation between the plates and the plates is less than 15%. Storage: 2-8 ° C, protected from light and moisture. Validity: 6 months Detection range: 1.25 ng/ml -80ng/ml Disclaimer The kit is for research use only and should not be used in clinical trials or rat body experiments. Otherwise, all the consequences will be borne by the experimenter. Not responsible. Strictly follow the instructions, different batch numbers can not be exchanged, the experimenter violates the instructions, and the consequences are borne by the experimenter.
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