In 1971, Engvall and Perlmann published an article on enzymelinked immunosorbent assay (ELISA) for the quantitative determination of IgG, which led to the development of enzyme-labeled antibody technology for antigen localization in 1966 to the determination of trace substances in liquid specimens. The basic principle of this method is: (1) to bind the antigen or antibody to the surface of a solid carrier and maintain its immune activity. (2) The antigen or antibody is linked to a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immune activity and the activity of the enzyme. During the assay, the tested specimen (the antibody or antigen in which it is measured) and the enzyme label antigen or antibody react with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid support is separated from other substances by washing, and then the amount of enzyme bound to the solid support is proportional to the amount of the tested substance in the specimen. After adding the substrate of the enzymatic reaction, the substrate is catalyzed by the enzyme into a colored product, and the amount of the product is directly related to the amount of the detected substance in the specimen, so it can be qualitatively or quantitatively analyzed according to the depth of the color reaction. Due to the high catalytic frequency of the enzyme, the reaction effect can be greatly amplified, resulting in a high sensitivity of the assay method.
Method type and procedure
ELISA can be used to determine antigens as well as antibodies. There are 3 necessary reagents in this assay: (1) antigens or antibodies in the solid phase, (2) enzyme-labeled antigens or antibodies, and (3) substrates for enzyme action. According to the source of the reagent, the characteristics of the specimen, and the conditions for detection, various different types of detection methods can be designed.
(A) Double antibody sandwich method
The double antibody sandwich method is a commonly used method for antigen detection, and the operation steps are as follows:
(1) Connect the specific antibody to the solid-phase carrier to form a solid-phase antibody: wash to remove the unbound antibody and impurities.
(2) Add the tested specimen: make it react with the solid-phase antibody for a period of time, so that the antigen in the specimen can combine with the antibody on the solid-phase carrier to form a solid-phase antigen complex. Wash to remove other unbound substances.
(3) Enzyme-labeled antibody: the antigen on the solid-phase immune complex is bound to the enzyme-labeled antibody. Wash the unconjugated enzyme-labeled antibody thoroughly. At this time, the amount of enzyme carried on the solid support is positively correlated with the amount of the substance being tested in the specimen.
(4) Substrate addition: The enzyme-catalyzed substrate in the sandwich complex becomes a colored product. Qualitative or quantitative of this antigen is performed depending on the degree of color reaction.
According to the same principle, the macromolecular antigen is prepared separately from the solid phase antigen and the enzyme label antigen conjugate, and the antibody in the specimen can be determined by the double antigen sandwich method.
(B) One-step method of two sites
In the determination of antigen by the double-antibody sandwich method, if the single antibody against two different epitopes on the antigen molecule is used as the solid-phase antibody and the enzyme-labeled antibody respectively, the addition of the specimen and the addition of the enzyme-labeled antibody can be made in two steps and one step in the determination. This two-site step not only simplifies the procedure and shortens the reaction time, but also significantly improves the sensitivity and specificity of the assay by using high-affinity single antibodies. The application of single antibodies has taken ELISA to a new level for the determination of antigens.
In the one-step assay, attention should be paid to the hook effect, which is similar to the posterior band phenomenon of excess antigen in the precipitation reaction. When the concentration of the antigen to be measured in the specimen is quite high, the excess antigen binds to the solid-phase antibody and the enzyme-labeled antibody respectively, and no longer forms a sandwich complex, and the obtained result will be lower than the actual content. In severe cases, false-negative results may even occur.
(C) Indirect detection of antibodies
The indirect method is a commonly used method for the detection of antibodies, and its principle is to use enzyme-labeled antibodies to detect the tested antibodies that have been bound to solids, so it is called indirect method. The procedure is as follows:
(1) Connect the specific antigen with the solid-phase carrier to form a solid-phase antigen: wash to remove the unbound antigen and impurities.
(2) Diluted tested serum: the specific antibody in it binds to the antigen to form a solid-phase antigen-antibody complex. After washing, only the specific antibody is left on the solid support. Other immunoglobulins and impurities in the serum are washed away during the washing process because they cannot bind to the solid-phase antigen.
(3) Enzyme-labeled 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, if you want to test human antibodies to a certain disease, you can use enzyme labeled sheep anti-human IgG antibodies.
(4) Substrate color development: The color depth represents the amount of antibody to be tested in the specimen.
In this method, as long as different solid-phase antigens are replaced, a enzyme-labeled anti-antibody can be used to detect various antibodies corresponding to the antigen.
(D) Competition Law
The competitive method can be used for the determination of antigens and can also be used for the determination of antibodies. For example, in the case of antigen determination, the tested antigen and the enzyme labeled antigen compete to bind to the solid-phase antibody, so the amount of enzyme-labeled antigen bound to the solid phase is inversely proportional to the amount of the antigen tested. The procedure is as follows:
(1) The specific antibody is connected to the solid-phase carrier to form a solid-phase antibody. Wash.
(2) Add a mixed solution of the tested specimen and a certain amount of enzyme labeled antigen to the tube to be tested, so that it reacts with the solid-phase antibody. If there is no antigen in the specimen, the enzyme label antigen can bind to the solid-phase antibody smoothly. If the test specimen contains antigen, it binds to the solid-phase antibody with the same chance as the enzyme-labeled antigen, which competitively takes up the opportunity for the enzyme-labeled antigen to bind to the solid-phase carrier, so that the binding amount of the enzyme-labeled antigen to the solid-phase carrier is reduced. Only the enzyme-labeled antigen is added to the reference tube, and after heat preservation, the binding of the enzyme-labeled antigen to the solid-phase antibody can reach a sufficient amount. Wash.
(3) Substrate color development: The color in the reference tube is darker due to the large number of bound enzyme label antigens. 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 being tested. The lighter the color of the tube to be measured, the more antigen content is in the specimen.
(E) IgM antibody detection by capture method
Specific IgM for certain antigens is often present in serum, and specific IgG is present, the latter interfering with the determination of IgM antibodies. Therefore, the IgM anti-IgM antibody is mostly determined by the capture method, in which all serum IgM (including heterosexual IgM and non-specific IgM) is fixed on a solid phase, and the specific IgM is determined after IgG is removed. The procedure is as follows:
(1) The anti-human IgM antibody is attached to the solid-phase support to form the solid-phase anti-human IgM. Wash.
(2) Add diluted serum specimens: IgM antibodies in the serum are captured by solid-phase antibodies after the holding reaction. Wash to remove other immunoglobulins and impurities from serum.
(3) Add a specific antigen reagent: it only binds to specific IgM on the solid phase. Wash.
(4) Add specific enzyme-labeled antibody: make it bind to the antigen reaction bound to the solid phase. Wash.
(5) Substrate color development: If there is a color display, it means that there are specific IgM antibodies in the serum specimen, which is a positive reaction.
(F) ELISA with avidin and biotin applied
Avidin is a glycoprotein that can be extracted from egg whites. The molecular weight is 60 kD, and each molecule is composed of 4 subunits, which can be intimately bound to 4 biotin molecules. More streptomycin and strepavidin extracted from Streptomyces are now used. Biotin, also known as vitamin H, has a molecular weight of 244.31 and is present in egg yolks. Biotin-hydroxysuccinimide (BNHS), a chemically made derivative, can form biotinylated products with various types of large and small molecules, such as proteins, sugars, and enzymes. The combination of avidin and biotin, although it is not an immune response, has strong specificity and affinity, and the two are extremely stable once combined. Since 1 avidin molecule has the binding position of 4 biotin molecules, more biotinylated molecules can be joined to form a lattice-like complex. Therefore, conjugating avidin and biotin to an ELISA can greatly increase the sensitivity of the ELISA.
The avidin-biotin system can be used in ELISA in a variety of forms, from indirect coating to final reaction scale-up. The antibody or antigen coated with the solid phase can be pre-coated on the solid phase first, and the antibody or antigen coated with the solid phase by adsorption method is combined with biotin, and the biotinylated antibody or anti-antibody is in-phase through the avidin-biotin reaction. This coating method not only increases the amount of adsorbed antibody or antigen, but also fully exposes the binding site. In addition, enzyme-labeled antibodies in conventional ELISAs can be replaced with biotinylated antibodies that are then ligated with avidin-enzyme conjugates to amplify the reaction signal.
ELISA is commonly used as a bonding assay for non-radioactive isotopes. In this method, usually the standard ligand is immobilized and bonded by the addition of a solution-phase acceptor or protein. Bonding receptors are quantified by the addition of an antibody that reacts specifically with the receptor, and the amount of the initial antibody is measured by the addition of a second chromogenic antibody. The second antibody recognizes the antibody
