Second, ELISA principle and classification
1. Principle of ELISA
The basis of ELISA is the immobilization of antigens or antibodies and the enzymatic labeling of antigens or antibodies. The antigen or antibody bound to the surface of the solid support retains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and the activity of the enzyme. At the time of measurement, the test specimen (the antibody or antigen in which it is measured) reacts with an antigen or antibody on the surface of the solid phase carrier. The antigen-antibody complex formed on the solid support is separated from other substances in the liquid by washing. Further, an enzyme-labeled antigen or antibody is added, and is also bound to the solid phase carrier by a reaction. At this time, the amount of enzyme on the solid phase is proportional to the amount of the substance to be tested in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of the test substance in the sample, so that qualitative or quantitative analysis can be performed according to the depth of the color. Due to the high catalytic efficiency of the enzyme, the result of the immune reaction is indirectly amplified, and the measurement method achieves high sensitivity.
2. Type of ELISA
ELISA can be used to determine antigens and can also be used to determine antibodies. There are three essential reagents in this assay: (1) solid phase antibiotics or antibodies, ie "immunosorbents"; (2) enzyme-labeled antigens or antibodies, called "conjugates" ( Conjugate); (3) substrate for the enzymatic reaction. Depending on the source of the reagent and the condition of the specimen and the specific conditions of the assay, various types of assays can be devised. The ELISAs used for clinical testing are mainly of the following types:
2.2.1 Double antibody sandwich assay for antigen
The double antibody sandwich method is the most commonly used method for detecting antigens. The steps are as follows:
1) A specific antibody is linked to a solid phase carrier to form a solid phase antibody. Washing removes unbound antibodies and impurities.
2) Add the specimen to be inspected and keep warm. The antigen in the specimen binds to the solid phase antibody to form a solid phase antigen-antibody complex. Wash to remove other unbound material.
3) Add the enzyme-labeled antibody and keep the reaction. The antigen on the solid phase immune complex binds to the enzyme-labeled antibody. Unbound enzyme-labeled antibody was thoroughly washed. The amount of enzyme carried on the solid support at this time is related to the amount of antigen to be detected in the specimen.
4) Add substrate to develop color. The enzyme on the solid phase catalyzes the substrate to become a colored product. The amount of antigen in the specimen is measured by colorimetry. In clinical tests, this method is suitable for testing macromolecular antigens such as various proteins such as HBsAg, HBeAg, AFP, hCG, and the like. This method can be established by coating a solid phase carrier and preparing an enzyme conjugate as long as the heterologous antibody against the test antigen is obtained. For example, the source of the antibody is an antiserum, and the antibodies for labeling and labeling are preferably taken from animals of different species. If monoclonal antibodies are used, two monoclonal antibodies directed against different determinants of the antigen are typically selected for coating the solid support and preparing the enzyme conjugate, respectively. The two-site sandwich method has high specificity, and the test specimen and the enzyme-labeled antibody can be incubated together for one-step detection.
2.2.2 Double antigen sandwich assay antibody
The reaction pattern is similar to the double antibody sandwich method. The enzyme conjugate is coated and prepared with a specific antigen to detect the corresponding antibody. The difference from the indirect method for measuring antibodies is to replace the enzyme-labeled anti-antibody with an enzyme-labeled antigen. In this method, the specimen to be inspected does not need to be diluted, and can be directly used for measurement, 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 to this method lies in the preparation of the enzyme-labeled antigen, and the appropriate labeling method should be found according to the difference in antigen structure.
2.2.3 Indirect method for measuring antibodies
The indirect method is a commonly used method for detecting antibodies. The principle is to use an enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect a test antibody that binds to a solid phase antigen, so it is called an indirect method (see Figure 2-3). The steps are as follows:
2.2.4 Competition Law Test
Specific antibodies can be detected by this method when interfering substances in the antigen material are not easily removed, or when it is difficult to obtain sufficient purified antigen. The principle is that the antibody in the specimen competes with a certain amount of the enzyme-labeled antibody for binding to the solid phase antigen. The more the amount of antibody in the specimen, the less the enzyme-labeled antibody bound to the solid phase, so the positive reaction was lighter than the negative reaction. If the antigen is of high purity, it can be directly coated with a solid phase. If there is an interfering substance in the antigen, the direct coating is not easy to be successful, and the capture coating method may be employed, that is, the antibody corresponding to the solid phase antigen is first coated, and then the antigen is added to form a solid phase antigen.
2.2.5 Competition method to measure antigen
Small molecule antigens or semi-antibiotics lack two or more sites that can be used as sandwich methods, and therefore cannot be measured by the double antibody sandwich method, and a competition method mode can be employed. The principle is that the antigen in the specimen competes with a certain amount of the enzyme-labeled antigen for binding to the solid phase antibody. The more the antigen content in the specimen, the less the enzyme-labeled antigen bound to the solid phase, and the final color development is shallower. This method is often used for ELISA assays such as small molecule hormones and drugs.
2.2.6 capture coating antibody
Detection of IgM antibodies is used in the early diagnosis of infectious diseases. Indirect ELISA is generally only suitable for detecting total antibodies or IgG antibodies. If the IgM antibody is directly assayed by an indirect method of antigen coating, a higher concentration of IgG antibody is generally present in the sample, and the latter will compete for binding to the solid phase antigen so that a part of the IgM antibody cannot bind to the solid phase. Therefore, if IgM antibodies are indirectly measured using anti-human IgM as a secondary antibody, the sample must first be treated with protein A or anti-IgG to remove IgG interference. The capture coating method is often used in the determination of antibody IgM in clinical tests. The solid phase is first coated with an anti-human IgM antibody to capture IgM in serum samples (including specific IgM antibodies to the antigen and non-specific IgM).
2.2.7 ABS-ELISA
ABS is an abbreviation of avidin biotin system. Avidin is a glycoprotein with a molecular weight of 60,000 and consists of four subunits that bind to biotin. Biotin is a small molecule compound with a molecular weight of 244. The chemically-derived derivative-hydroxysuccinimide ester can form biotin-labeled products with various types of large and small molecules such as proteins and sugars, and the labeling method is quite simple.
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