ELISA， Enzyme linked immunosorbent assay (ELISA) refers to the qualitative and quantitative method of adsorbing soluble antigens or antibodies onto a solid-phase carrier for immune reactions. In 1971, Engvall and Perlmann published an article on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of IgG, which led to the development of enzyme-linked antibody technology for antigen localization into a method for detecting trace substances in liquid specimens starting in 1966. ELISA can be used to detect antigens as well as antibodies. There are three necessary reagents in this measurement method: ① solid-phase antigen or antibody, ② enzyme labeled antigen or antibody, and ③ substrate for enzyme action. Based on the source of reagents, the characteristics of specimens, and the conditions for detection, various types of detection methods can be designed.
1. Double antibody sandwich method
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) Add substrate: The enzyme in the sandwich complex catalyzes the substrate to become a colored product. 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.
2. Double site one-step 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 approach 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.
3. Indirect method for detecting antibodies
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, indirectly 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.
4. Competitive testing of antibodies
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. wash.
(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. wash.
(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.
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