Fish anti-liver kidney microsomal antibody (LKM) ELISA kit detection principle kit using double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells in which the fish anti-liver kidney microsomal antibody (LKM) was pre-coated, the specimen, the standard, and the HRP-labeled detection antibody were sequentially added, and the cells were washed 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 fish anti-liver kidney microsomal antibodies (LKM) 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 Â°C, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly. Self-prepared items 1. Microplate reader (450nm) 2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL3. 37Â°C incubator operation precautions 1. Kit storage At 2-8 Â° C, equilibrate for 20 minutes at room temperature before use. 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. 2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage. 3. The pre-treated sample does not need to be diluted, and 10 Î¼L of the sample can be directly added. 4. Incubate the operation strictly in accordance with the time indicated in the instructions, the amount of liquid added, and the sequence. 5. Shake well all liquid components before use. Kit Composition Name 96-well configuration 48-well configuration Remarks Microporous microplates 12 wells Ã— 8 strips 12 wells Ã— 4 no standards (2000 pg/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 Ã— 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: 2000, 1000, 500, 250, 125, 0pg/ml. Preparation of reagents 20Ã— Dilution of Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20X wash buffer plus 19 parts of distilled water. Washing method 1. Manually wash the plate: Drain 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. 2. Automatic washing machine: Inject 350Î¼L of washing solution into each hole, soak for 1min, and wash the plate 5 times. Procedure 1. Remove the required slats from the foil pouch after equilibrating for 20 min at room temperature. The remaining slats were sealed back to 4 Â°C with a ziplock bag. 2. Set standard hole, sample hole and blank hole, blank hole is added; standard product hole is added with different concentration of standard product 50Î¼L; 3. Sample hole to be tested first add 10Î¼L sample to be tested, plus sample diluent 40 Î¼L; 4. Then add 50 Î¼L of horseradish peroxidase (HRP)-labeled detection antibody to the standard well and sample well (no blank), seal the well with a sealing membrane, 37 Â° C water bath or constant temperature The box was incubated for 60 min. 5. 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). 6. Add 50 Î¼L of substrate A and B to all wells and incubate for 15 min at 37 Â° C in the dark. 7. Add 50 Î¼L of stop solution to all wells and measure the OD value of each well 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 â€‹â€‹are calculated according to the curve equation. Kit Performance 1. Accuracy: The linear regression coefficient of the standard and the expected concentration correlation coefficient R value, greater than or equal to 0.9900. 2. Sensitivity: The minimum detection concentration is less than 1.0 pg/ml. 3. Specificity: Does not cross-react with other soluble structural analogs. 4. Repeatability: The coefficient of variation between the plates and the plates is less than 15%. 5. Storage: 2-8 Â° C, protected from light and moisture. 6. Validity: 6 months 7. Detection range: 31.2pg/ml-2000pg/ml Disclaimer 1. The kit is for research use only and should not be used in clinical trials or fish experiments, otherwise all consequences will be determined by the experiment. The company assumes that the company is not responsible. 2. Strictly follow the instructions. Different batch numbers cannot be exchanged. The experimenter violates the instructions and the consequences are borne by the experimenter.
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