Fundamental
In 1971, Engvall and Perlmann published the article of enzyme linked immunosorbent assay (ELISA) for quantitative determination of IgG, making the enzyme-labeled antibody technology for antigen localization developed in 1966 into trace substances in liquid samples. test methods.
The basic principle of this method is:
â‘ Make the antigen or antibody bind to the surface of a solid carrier and maintain its immunological activity.
â‘¡ The antigen or antibody is linked to an enzyme to form an enzyme-labeled antigen or antibody. This enzyme-labeled antigen or antibody retains both its immunological activity and enzyme activity. During the measurement, the test specimen (the antibody or antigen therein) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the amount of enzyme bound on the solid phase carrier is in a certain proportion to the amount of the tested substance in the specimen. After adding the substrate for the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the test substance in the specimen, so it can be qualitatively or quantitatively analyzed according to the depth of the color reaction.
Since the catalytic frequency of the enzyme is very high, the reaction effect can be greatly amplified, so that the measurement method reaches a high sensitivity. Method types and operating procedures ELISA can be used to determine antigens as well as antibodies.
There are three necessary reagents in this measurement method: â‘ solid-phase antigen or antibody, â‘¡ enzyme-labeled antigen or antibody, â‘¢ substrate for enzyme action. According to the source of the reagent and the properties of the specimen and the conditions for the 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. The steps are as follows:
(1) Connect a specific antibody to a solid phase carrier to form a solid phase antibody: wash to remove unbound antibody and impurities.
(2) Add the test specimen: make it contact with the solid-phase antibody and react for a period of time to allow the antigen in the specimen to combine with the antibody on the solid-phase carrier to form a solid-phase antigen complex. Wash to remove other unbound materials.
(3) Adding enzyme-labeled antibody: the antigen on the solid-phase immune complex is combined with the enzyme-labeled antibody. Wash unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme carried on the solid phase carrier is positively correlated with the amount of the tested substance in the specimen.
(4) Substrate addition: The enzyme in the sandwich compound catalyzes the substrate to become a colored product. Qualitative or quantitative determination of the antigen is carried out according to the degree of color reaction. According to the same principle, the solid antigen and enzyme-labeled antigen conjugate are prepared separately by the macromolecular antigen, and the antibody in the specimen can be determined by the double antigen sandwich method.
(2) Two-site one-step method When the antigen is measured by the double antibody sandwich method, if monoclonal antibodies against two different antigenic determinants on the antigen molecule are used as solid-phase antibodies and enzyme-labeled antibodies, respectively, the specimen can be used for the measurement And the enzyme-labeled antibody are added in two steps (Figure 15-5). This dual site not only simplifies the operation and shortens the reaction time, such as the use of high-affinity monoclonal antibodies, the sensitivity and specificity of the assay are also significantly improved. The application of monoclonal antibodies has brought the ELISA for antigen determination to a new level.
In the one-step determination, attention should be paid to the hook effect, which is similar to the post-band phenomenon of excess antigen in the precipitation reaction. When the concentration of the antigen to be tested in the specimen is quite high, the excess antigen will bind to the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a sandwich complex, and the result will be lower than the actual content. False negative results can even occur when the hook effect is severe.
(3) Indirect method for measuring 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 test antibody that has been bound to the solid phase, so it is called the indirect method. The operation steps are as follows:
(1) Connect the specific antigen to the solid phase carrier to form a solid phase antigen: wash to remove unbound antigen and impurities.
(2) Diluted test serum: the specific antibody in it binds to the antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies are left on the solid phase carrier. Impurities in other immunoglobulins and serum cannot be combined with solid-phase antigens and are washed away during the washing process.
(3) Add enzyme-labeled anti-antibody: bind to the antibody in the solid phase complex, so that the antibody is indirectly labeled with the enzyme. After washing, the amount of enzyme on the solid support represents the amount of specific antibody. For example, to test human antibodies to a disease, enzyme-labeled goat anti-human IgG antibodies can be used.
(4) Color development by adding substrate: the color depth represents the amount of antibody tested in the specimen. In this method, as long as different solid phase antigens are replaced, an enzyme-labeled anti-antibody can be used to detect various antibodies corresponding to the antigen.
(4) Competition method The competition method can be used to determine antigens and antibodies. Taking the measurement of antigen as an example, the test antigen and the enzyme-labeled antigen compete for binding with the solid-phase antibody, so the amount of the enzyme-labeled antigen bound to the solid phase is inversely proportional to the amount of the tested antigen. The operation steps are as follows:
(1) The specific antibody is connected to the solid phase carrier to form a solid phase antibody. washing.
(2) Add a mixed solution of the test specimen and a certain amount of enzyme-labeled antigen to the test tube to make it react with the solid phase antibody. If there is no antigen in the tested specimen, the enzyme-labeled antigen can smoothly bind to the solid-phase antibody. If the test specimen contains antigen, it will bind to the solid-phase antibody at the same opportunity as the enzyme-labeled antigen, competitively taking up the opportunity for the enzyme-labeled antigen to bind to the solid-phase carrier, so that the enzyme-labeled antigen will bind to the solid-phase carrier The amount is reduced. Only the enzyme-labeled antigen is added to the reference tube. After incubation, the enzyme-labeled antigen and the solid-phase antibody can be combined to the fullest amount. washing.
(3) Color development by adding substrate: the reference tube contains the most enzyme-labeled antigen, so the color is the darkest. The difference between the color depth of the reference tube and the color depth of the tube to be tested represents the amount of antigen in the specimen under test. The lighter the tube to be tested, the more the antigen content in the specimen.
(5) Measurement of IgM antibody by capture method
The specific IgM for certain antigens in serum often coexists with specific IgG, which can interfere with the determination of IgM antibodies. Therefore, the multi-capture method is used to determine IgM resistance. First, all serum IgM (including heterogeneous IgM and non-specific IgM) is fixed on the solid phase, and the specific IgM is determined after removing IgG.
The operation steps are as follows:
(1) Connect the anti-human IgM antibody to the solid phase carrier to form a solid phase anti-human IgM. washing.
(2) Add diluted serum specimens: IgM antibody in serum is captured by solid phase antibody after incubation reaction. Wash to remove impurities in other immunoglobulins and serum.
(3) Add specific antigen reagent: it only binds to specific IgM on the solid phase. washing.
(4) Add enzyme-specific antibody for specificity: make it react with and bind to the antigen bound on the solid phase. washing.
(5) Color development by adding substrate: if the color is displayed, it means that the specific IgM antibody in the serum sample is present, which is a positive reaction.
(6) ELISA using avidin and biotin Avidin is a glycoprotein that can be extracted from egg white. With a molecular weight of 60kD, each molecule is composed of 4 subunits and can be intimately combined with 4 biotin molecules. Now more streptavidin is extracted from Streptomyces. Biotin (also known as vitamin H) has a molecular weight of 244.31 and is present in egg yolk. Derivatives made by chemical methods, biotin-hydroxysuccinimide (BNHS) can form biotinylated products with various types of large and small molecules such as proteins, sugars and enzymes. Although the combination of avidin and biotin is not an immune response, it has strong specificity and high affinity, and the two are extremely stable once combined. Since one avidin molecule has four biotin molecule binding positions, more biotinylated molecules can be connected to form a lattice-like complex. Therefore, coupling avidin and biotin with ELIS can greatly improve the sensitivity of ELISA.
The avidin-biotin system can be used in ELISA in various forms. It can be used for indirect coating and can also be used for final reaction amplification. The avidin can be pre-coated on the solid phase. The antibody or antigen coated on the solid phase by the adsorption method is combined with biotin, and the biotinylated antibody or anti-phase can be phased by the avidin-biotin reaction. . This coating method can not only increase the amount of adsorbed antibody or antigen, but also fully expose its binding point. In addition, the enzyme-labeled antibody in the conventional ELISA can also be replaced with a biotinylated antibody, and then the avidin-enzyme conjugate is connected to amplify the reaction signal. ELISA is commonly used as a bonding assay for non-radioactive isotopes. In this method, the standard ligand is usually immobilized by adding solution phase receptors or proteins to bond them. By adding antibodies that specifically react with the receptor To quantify the bound receptors, and the amount of the initial antibody is measured by adding a second color-developing antibody. The second antibody recognizes the end of the antibody, alkaline phosphate or peroxidase at its end The enzyme reacts, which causes the solution to develop color.
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