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Introduction of the double antibody sandwich method

Views : 1668
Update time : 2023-03-01 11:44:51
The double antibody sandwich method is suitable for the determination of large molecule antigens that are bivalent or above, but not for the determination of semi-antigens and small molecule monovalent antigens, as they cannot form a two-dot sandwich. Today I will talk to you about why the double antibody sandwich method is a common method for the detection of antigens?
1. The specific antibody is conjugated to a solid phase carrier to form a solid phase antibody. Wash to remove unbound antibodies and impurities.
2、Add the specimen under test and hold the reaction. The antigen in the specimen binds to the solid phase antibody to form a solid phase antigen-antibody complex. Washing to remove other unbound material.
3、Add enzyme-labeled antibodies, holding reaction. The antigen on the solid phase immune complex binds to the enzyme-labeled antibody. Thoroughly wash the unbound enzyme-labeled antibodies. At this point, the amount of enzyme on the solid phase carrier is related to the amount of antigen detected in the specimen.
4. Add substrate for color development. The enzyme on the solid phase catalyzes the substrate to become a colored product. By colorimetric, the amount of antigen in the specimen is measured.
In clinical testing, this method is suitable for testing various proteins and other large molecule antigens, such as HBsAg, HBeAg, AFP, hCG, etc. As long as heterogeneous antibodies are obtained against the antigen under test, the method can be established by encapsulating the solid phase carrier and preparing enzyme conjugates. If the source of the antibody is antiserum, the antibodies for encapsulation and enzyme labeling are taken from different species of animals, respectively. In the case of monoclonal antibodies, two monoclonal antibodies against different determinants of the antigen are generally selected for the encapsulation of the solid phase vector and the preparation of the enzyme conjugate, respectively. This two-site sandwich method has high specificity and allows for a one-step assay by holding the specimen under test and the enzyme-labeled antibody together. In the one-step assay, when the specimen contains high levels of the tested antigen, the excess antigen binds to the solid phase antibody and the enzyme-labeled antibody, respectively, and no more sandwich complexes are formed. The absorbance value of the reaction is the same as the absorbance value of the standard curve (located on the antibody excess band) for a certain antigen concentration.
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