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Experimental principle and operation steps of human interleukin 6 kit
Experimental principle
This kit uses the double antibody sandwich method to determine the level of human interleukin 6 (IL-6) in the specimen. The microplate was coated with purified human interleukin 6 (IL-6) antibody to make a solid-phase antibody. Interleukin 6 (IL-6) was added to the monoclonal antibody-coated microwells in turn, followed by HRP labeled interleukin 6 IL-6) The antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with interleukin 6 (IL-6) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human interleukin 6 (IL-6) in the sample was calculated by a standard curve.
Steps
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.
8 ng / L
Standard No. 5
150μl of original standard is added to 150μl standard dilution
4 ng / L
Standard 4
150μl Standard No. 5 is added to 150μl standard dilution
2 ng / L
Standard 3
150μl Standard No. 4 is added to 150μl Standard Diluent
1 ng / L
Standard No. 2
150μl Standard No. 3 is added to 150μl Standard Diluent
0.5 ng / L
Standard 1
150μl Standard No. 2 is added to 150μl Standard Diluent
2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: dilute 20 times concentrated washing liquid with distilled water 20 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
Measurement: The absorbance (OD value) of each well was measured in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Detection range: 96T
0-8 ng / L
Note: This kit is used to determine the content of interleukin 6 (IL-6) in human serum, plasma and related liquid samples for scientific research use only.