This kit can only be used for scientific research. It should not be used for medical diagnosis. Human Matrix Metalloproteinase 13 (MMP-13) ELISA Kit The detection principle kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with human matrix metalloproteinase 13 (MMP-13) capture antibody, specimens, standards, HRP-labeled detection antibodies were sequentially added, 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 human matrix metalloproteinase 13 (MMP-13) 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 50 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 standard (6 tubes) 0.5 ml / tube 0.5 mL / tube without sample dilution 6 mL 3 mL No detection antibody -HRP 10mL 5mL No 20Ã— Wash Buffer 25mL 15mL Diluted substrate A 6mL 3mL without substrate B 6mL 3mL without stop solution 6mL 3mL without sealing film 2 sheets 2 sheets without instructions 1 part 1 without zipper bag 1 1 no note: 1. The concentration of the standard is: 640, 320, 160, 80, 40, 20 ng/mL. 2. If the sample value exceeds the highest concentration of the standard, the sample may be diluted appropriately with the sample diluent. . Preparation of reagents 20Ã— Wash buffer dilution: Distilled water was diluted 1:20, ie 1 part of 20Ã— 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, no blank hole is added; standard product hole is added with different concentration of standard product 50Î¼L; 3. Sample hole to be tested is added with 50Î¼L sample to be tested; 4. Subsequent standard 100 Î¼L 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 Â° C water bath or incubator. 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 and standard concentration correlation coefficient R value of the standard is greater than or equal to 0.9900. 2. Sensitivity: The minimum detection concentration is less than 1.0 ng/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 Disclaimer 1. The kit is for research use only and should not be used for clinical trials or human experiments. Otherwise, all consequences will be borne by the experimenter and the company will not be responsible. 2. Operate in strict accordance with the instructions, the experimenter violates the instructions, and the consequences are borne by the experimenter.
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