1. Double antibody sandwich assay for antigen
The double antibody sandwich method is the most commonly used method for antigen detection. The steps are as follows:
1) Connect specific antibodies to solid phase carriers to form solid phase antibodies. Wash to remove unbound antibodies and impurities.
2) Add the specimen to be tested and keep it warm. The antigen in the specimen combines with the solid-phase antibody to form a solid-phase antigen-antibody complex. Wash to remove other unbound materials.
3) Add enzyme-labeled antibody and incubate the reaction. The antigen on the solid-phase immune complex binds to the enzyme-labeled antibody. Wash unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme carried on the solid-phase carrier is related to the amount of tested antigen in the specimen.
4) Add substrate to develop color. The enzyme on the solid phase catalyzes the substrate into a colored product. By colorimetry, measure the amount of antigen in the specimen. In clinical testing, this method is suitable for testing large protein antigens such as various proteins, such as HBsAg, HBeAg, AFP, hCG, etc. As long as the heteroantibodies against the test antigen are obtained, it can be used to coat the solid phase carrier and prepare the enzyme conjugate to establish this method. If the source of the antibody is antiserum, the antibody used for coating and enzyme labeling is preferably obtained from animals of different species. If monoclonal antibodies are used, two monoclonal antibodies against different determinants on the antigen are generally selected for coating the solid phase carrier and preparing the enzyme conjugate. This two-site sandwich method has a high specificity, and can incubate the test specimen and enzyme-labeled antibody together for one-step detection.
In the one-step measurement, when the content of the tested antigen in the specimen is high, the excess antigen is bound to the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a "sandwich complex". Similar to the phenomenon of post-banding of excess antigen in the precipitation reaction, the absorbance value of the color developed after the reaction (located on the band of antigen excess) and the standard curve (located on the band of antibody excess) of a certain antigen concentration, such as Normally, the result obtained will be lower than the actual content. This phenomenon is called the hook effect, because the standard curve reaches a peak and bends like a hook. When the hook effect is serious, the reaction may not even develop color and a false negative result may appear. Therefore, when using a one-step reagent to determine the abnormally high content of the sample (such as HBsAg, AFP in serum and hCG in urine), the highest value of the measurable range should be noted. Preparation of such reagents with high affinity monoclonal antibodies can weaken the hook effect.
If the test molecule contains multiple identical determinants, such as the a determinant of HBsAg, the same monoclonal antibody for this determination can be used to coat the solid phase and prepare the enzyme conjugate. However, in the detection of HBsAg, attention should be paid to the subtype problem. HBsAg has four subtypes: adr, adw, ayr, and ayw. Although each subtype has the same a determinant reactivity, this is also the application of sandwich antibody Note the problem.
Another note of the double antibody sandwich method for antigen detection is the interference of rheumatoid factor (RF). RF is an autoantibody, mostly IgM type, which can bind to the Fc segment of various animal IgG. If the serum sample used for the detection of the double antibody sandwich method contains RF, it can serve as an antigen component and at the same time combine with the solid-phase antibody and the enzyme-labeled antibody, showing a false positive reaction. The F (ab) or Fab fragment is used as the enzyme conjugate reagent, because the Fc segment is removed, thereby eliminating RF interference. Whether the double-antibody sandwich ELISA reagent is affected by RF has been listed as an evaluation index for this type of reagent (). +) $ oy]
The double antibody sandwich method is suitable for the determination of bivalent or more bivalent large-molecule antigens, but it is not suitable for the determination of hapten and small-molecule monovalent antigens because it cannot form a two-point sandwich.
2. Double antigen sandwich assay for antibody
The reaction mode is similar to the double antibody sandwich method. Coating with specific antigens and preparation of enzyme conjugates to detect the corresponding antibodies. The difference with the indirect method is to use enzyme-labeled antigen instead of enzyme-labeled anti-antibody. In this method, the test specimen does not need to be diluted, and can be directly used for determination, so its sensitivity is relatively higher than the indirect method. This method is often used for the detection of anti-HBs in hepatitis B markers. The key of this method lies in the preparation of enzyme-labeled antigen, and the appropriate labeling method should be searched according to the structure of the antigen.
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