Determination of human adiponectin content by double antibody sandwich method - Master's thesis - Dissertation

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Adiponectin (ADP) is a hormone-like protein secreted by adipocytes, which has biological effects such as increasing insulin sensitivity, anti-diabetes and anti-atherosclerosis, and anti-inflammatory. Shanghai Jinma Bio-Providing Adiponectin elisa kit is only for scientific research and quantitatively detects the content of human adiponectin (ADP) in cell liquid, body fluid, tissue, serum and plasma.
The human adiponectin Elisa kit measures the level of human adiponectin (ADP) using a double antibody sandwich method. The microporous plate was coated with purified human adiponectin (ADP) antibody to prepare a solid phase carrier. In the experiment, samples and standards were sequentially added to the microplate, and HRP-labeled ADP antibody was added to form antibody-antigen- The enzyme-labeled antibody complex was repeatedly washed and added to the substrate TMB for color development. TMB is converted to blue under the catalysis of peroxidase, and the reaction is terminated with sulfuric acid to convert to yellow. The color depth is positively correlated with the ADP content in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader, and the ADP concentration in the test sample was calculated from the standard curve.
Human adiponectin detection kit steps:
1. Indirect method The indirect method of antibody is a commonly used method for detecting antibodies. The principle is to use an enzyme-labeled anti-antibody (secondary antibody) to detect a test antibody that binds to a solid phase antigen, so it is called an indirect method. The steps are as follows:
1. Linking a specific antigen to a solid phase carrier to form a solid phase antigen, washing to remove unbound antigen and impurities.
2. Add the diluted sample and keep the reaction. The specific antibody in the sample binds to the solid phase antigen to form a solid phase antigen-antibody complex. After washing, only the specific antibody remains on the solid support, and the other components in the sample are washed away during the washing process.
3. Add enzyme-labeled anti-antibody. The antibody in the solid phase immune complex binds to the enzyme-labeled antibody, thereby indirectly labeling the enzyme. After washing, the amount of enzyme on the solid support is positively correlated with the amount of antibody being tested in the sample.
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