Mouse interleukin 1β (IL-1β) ELISA kit operation method

Detection range: 96T2.5ng/L-80ng/L Use purpose: This kit is used to determine the content of interleukin-1β (IL-1β) in serum, plasma and related liquid samples. Experimental principle This kit uses the double antibody sandwich method to determine the level of mouse interleukin 1β (IL-1β) in the specimen. The microporous plate was coated with purified mouse interleukin 1β (IL-1β) antibody to prepare a solid phase antibody, and interleukin 1β (IL-1β) was sequentially added to the microcapsule of the coated monoclonal antibody, followed by HRP-labeled interleukin-1β. The (IL-1β) 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 interleukin 1β (IL-1β) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of mouse interleukin 1β (IL-1β) 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 (160ng / L) 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. 80ng/L No.5 standard 150μl original standard added 150μl standard dilution 40ng/L No.4 standard 150μl No.5 standard added 150μl standard dilution 20ng/L No.3 standard 150μl No.4 standard Add 150 μl standard dilution 10 ng/L No. 2 standard 150 μl No. 3 standard Add 150 μl standard dilution 5 ng/L No. 1 standard 150 μl No. 2 standard Add 150 μl 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. Liquor: 20 times concentrated washing solution diluted with distilled water 20 times and spare. 5. Wash: carefully remove the sealing film, discard the liquid, dry, fill each well with washing liquid, let stand for 30 seconds and then discard , 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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