The double antibody sandwich method is the most commonly used method for detecting antigens, and the operating 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 test specimen: Allow it to react with the solid-phase antibody for a period of time, allowing the antigen in the specimen to bind with the antibody on the solid-phase carrier, forming a solid-phase antigen complex. Wash to remove other unbound substances.

(3) Add enzyme-linked antibody: bind the antigen on the solid-phase immune complex to the enzyme-linked antibody. Thoroughly wash unbound enzyme-linked antibodies. At this point, the amount of enzyme carried on the solid-phase carrier is positively correlated with the amount of the test substance in the specimen.

(4) Substrate addition: Enzymes in sandwich complexes catalyze the substrate into colored products. Qualify or quantify the antigen based on the degree of color reaction.

According to the same principle, solid-phase antigen and enzyme-linked antigen complexes can be prepared separately from macromolecular antigens, which can be used to determine antibodies in specimens using the double antigen sandwich method.

When using the double antibody sandwich method to determine antigens, if monoclonal antibodies targeting two different antigenic determinants on the antigen molecule are used as solid-phase antibodies and enzyme-linked antibodies, the addition of the specimen and enzyme-linked antibody can be done in two steps and in one step. This dual site one-step method not only simplifies the operation and shortens the reaction time, but also significantly improves the sensitivity and specificity of the assay when using high affinity monoclonal antibodies. The application of monoclonal antibodies has elevated the ELISA for antigen detection to a new level.

In one-step determination, attention should be paid to the hook effect, similar to the phenomenon of antigen excess in the back band of precipitation reactions. When the concentration of the antigen to be tested in the specimen is relatively high, excess antigen will bind to solid-phase antibody and enzyme-linked antibody separately, instead of forming a sandwich complex, and the obtained result will be lower than the actual content. When the hook effect is severe, false negative results may even occur.

The indirect method is the most commonly used method for detecting antibodies. Its principle is to use enzyme labeled anti antibodies to detect the tested antibodies that have bound to the solid phase, hence it is called the indirect method. The operation steps are as follows:

(1) Connect specific antigens to solid-phase carriers to form solid-phase antigens: wash to remove unbound antigens and impurities.

(2) Diluted test serum: The specific antibodies in it bind to the antigen to form a solid-phase antigen antibody complex. After washing, only specific antibodies remain on the solid-phase carrier. Other immunoglobulins and impurities in serum are washed away during the washing process because they cannot bind to solid-phase antigens.

(3) Enzyme labeled anti antibody: binds to the antibody in the solid-phase complex, thereby labeling the antibody with an enzyme. After washing, the amount of enzyme on the solid-phase carrier represents the amount of specific antibody. For example, to test a person's antibodies against a certain disease, enzyme-linked sheep anti human IgG antibodies can be used.

(4) Color development with substrate: The color depth represents the amount of tested antibody in the specimen.

This method can detect various antibodies corresponding to antigens using a single enzyme-linked immunosorbent assay (ELISA) by replacing different solid-phase antigens.

Competition law can be used to determine antigens as well as antibodies. Taking the determination of antigens as an example, the tested antigen and enzyme-linked antigen compete for binding with solid-phase antibodies, so the amount of enzyme-linked antigen bound to the solid-phase is inversely proportional to the amount of tested antigen. The operation steps are as follows：

(1) Connect specific antibodies to solid-phase carriers to form solid-phase antibodies.

(2) Add a mixed solution of the test specimen and a certain amount of enzyme-linked antigen to the test tube to react with the solid-phase antibody. If there is no antigen in the tested sample, the enzyme-linked antigen can bind smoothly to the solid-phase antibody. If the tested specimen contains antigens, it will bind to the solid-phase antibody with the same chance as the enzyme-linked antigen, competitively occupying the opportunity for the enzyme-linked antigen to bind to the solid-phase carrier, reducing the binding amount between the enzyme-linked antigen and the solid-phase carrier. Only enzyme-linked antigen is added to the reference tube, and after insulation, the binding between enzyme-linked antigen and solid-phase antibody can reach the maximum amount.

(3) Color development with substrate addition: The reference tube has the darkest color due to the highest number of bound enzyme-linked antigens. The difference between the color depth of the reference tube and the color depth of the test tube represents the amount of antigen in the specimen being tested. The lighter the color of the test tube, the higher the antigen content in the specimen.

血Specific IgM for certain antigens in Qingzhong often coexists with specific IgG, which can interfere with the determination of IgM antibodies. Therefore, the capture method is often used to determine IgM antibodies. First, all serum IgM (including specific IgM and non-specific IgM) is fixed on a solid phase, and after removing IgG, specific IgM is measured. The operation steps are as follows:

(1) Connect the anti human IgM antibody onto a solid-phase carrier to form solid-phase anti human IgM.

(2) Add diluted serum sample: After the incubation reaction, IgM antibodies in the serum are captured by solid-phase antibodies. Wash to remove impurities from other immunoglobulins and serum.

(3) Add specific antigen reagent: It only binds to specific IgM on the solid phase.

(4) Add enzyme labeled antibodies targeting specificity: react and bind with antigens bound to the solid phase.

(5) Substrate color development: If there is a color display, it indicates the presence of specific IgM antibodies in the serum sample, which is a positive reaction.

Affinity protein is a glycoprotein that can be extracted from egg white. The molecular weight is 60kD, and each molecule is composed of 4 subunits, which can intimately bind to 4 biotin molecules. Nowadays, more streptavidin is extracted from Streptomyces. Biotin, also known as vitamin H, has a molecular weight of 244.31 and is found in egg yolks. A derivative made by chemical methods, biotin hydroxysuccinimide ester (BNHS), can form biotinylated products with various types of large and small molecules such as proteins, sugars, and enzymes. The binding of avidin and biotin, although not an immune response, has strong specificity and affinity, and the two become extremely stable once bound. Due to the fact that one avidin molecule has four binding sites for biotin molecules, it can connect more biotinylated molecules to form a lattice like complex. Therefore, coupling avidin and biotin with ELISA can greatly improve the sensitivity of ELISA.

The application of avidin biotin system in ELISA has various forms, including indirect encapsulation and final reaction amplification. It is possible to pre coat with avidin on the solid phase, and the antibody or antigen originally coated on the solid phase by adsorption can bind with biotin. The biotinylated antibody or antibody can be immobilized through the avidin biotin reaction. This coating method not only increases the amount of adsorbed antibodies or antigens, but also fully exposes their binding sites. In addition, enzyme-linked antibodies in conventional ELISA can also be replaced with biotinylated antibodies, and then linked to affinity enzyme complexes to amplify the reaction signal.

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