Detection range: 96T0.6ng/ml - 16ng/ml Use purpose: This kit is used to determine the content of integrin-like metalloproteinase 12 (ADAM12) in human serum, plasma and related liquid samples. Experimental principle This kit uses the double antibody sandwich method to determine the level of human integrin-like metalloproteinase 12 (ADAM12) in the specimen. The microporous
Plate was coated with purified human integrin-like metalloproteinase 12 (ADAM12) antibody to prepare a solid phase antibody, and the integrin-like metalloproteinase 12 (ADAM12) was sequentially added to the microcapsule of the coated monoclonal antibody, and then The HRP-labeled disintegrin-like metalloproteinase 12 (ADAM12) antibody binds to form 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 the disintegrin-like metalloproteinase 12 (ADAM12) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human integrin-like metalloproteinase 12 (ADAM12) in the sample was calculated from a standard curve. Kit composition 1 30 times concentrated washing solution 20ml × 1 bottle 7 Stop solution 6ml × 1 bottle 2 Enzyme standard reagent 6ml × 1 bottle 8 standard product (32ng / ml) 0.5ml × 1 bottle 3 enzyme label coating plate 12 holes × 8 9 standard dilutions 1.5ml × 1 bottle 4 sample dilution 6ml × 1 bottle 10 instructions 1 part 5 color reagent A liquid 6ml × 1 bottle 11 sealing film 2 sheets 6 coloring agent B liquid 6ml × 1 Bottle 12 sealed bag 1 specimen requirement 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 the standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart. 16ng/ml No.5 standard 150μl original standard added 150μl standard dilution 8ng/ml No.4 standard 150μl No.5 standard Add 150μl standard dilution 4ng/ml No.3 standard 150μl No.4 standard Add 150μl standard dilution 2ng/ml No. 2 standard 150μl No. 3 standard Add 150μl standard dilution 1ng/ml No. 1 standard 150μl No. 2 standard Add 150μl standard dilution 2. Add sample: Blank holes are respectively set (the blank control hole is not added with the sample and the enzyme standard reagent, and the other steps are the same), the standard hole, and the sample hole 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. 3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes. 4. Solution: Dilute 30 times concentrated washing solution with distilled water 30 times and reserve. 5. Wash: carefully remove the sealing film, discard the liquid, dry it, fill each well with washing liquid, let stand for 30 seconds and discard it. , repeat this 5 times, pat dry. 6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: add 50 μl of color developer A, add 50 μl of color developer B, gently shake and mix, and avoid light at 37 °C. 15 minutes. 10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned 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.
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