Principles and types of ELISA

1. The principle of ELISA

The basis of ELISA is the immobilization of antigen or antibody and the enzyme labeling of antigen or antibody. The antigen or antibody bound to the surface of the solid phase carrier still maintains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and enzyme activity. During the measurement, the test specimen (the antibody or antigen in the measurement) reacts with the antigen or antibody on the surface of the solid-phase carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. After adding enzyme-labeled antigen or antibody, it is also bound to the solid-phase carrier through the reaction. At this time, the amount of enzyme on the solid phase is proportional to the amount of test substance in the specimen. After adding the substrate of 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 qualitative or quantitative analysis can be carried out according to the intensity of the color. Due to the high catalytic efficiency of the enzyme, it indirectly amplifies the result of the immune response and makes the determination method reach a high sensitivity.

2. Types of ELISA

ELISA can be used to determine antigens and antibodies. There are three necessary reagents in this determination method:

(1) Antigen or antibody in solid phase, namely “immunosorbent” (immunosorbent);

(2) Enzyme-labeled antigens or antibodies are called “enzyme conjugates” or “conjugates”;

(3) Substrate of enzyme reaction. According to the source of the reagent and the condition of the specimen and the specific conditions of the test, various different types of detection methods can be designed. There are mainly the following types of ELISA for clinical testing:

(1) 　Double antibody sandwich method for antigen detection

The double antibody sandwich method is the most commonly used method for detecting antigens. The operation steps are as follows:

(1) Connect the specific antibody with the solid-phase carrier to form a solid-phase antibody. Wash to remove unbound antibody and impurities.

(2) Add the tested specimen and keep warm for reaction. The antigen in the specimen combines with the solid-phase antibody to form a solid-phase antigen-antibody complex. Wash to remove other unbound substances.

(3) Add enzyme-labeled antibody and incubate the reaction. The antigen on the solid-phase immune complex binds to the enzyme-labeled antibody. Wash the unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme carried on the solid phase carrier is related to the amount of the tested antigen in the specimen.

(4) Add substrate to develop color. The enzyme on the solid phase catalyzes the substrate to become a colored product. Through colorimetry, the amount of antigen in the specimen is measured.

In clinical testing, this method is suitable for testing various proteins and other macromolecular antigens, such as HBsAg, HBeAg, AFP, hCG, etc. As long as the heterogeneous antibody against the tested antigen is obtained, it can be used to coat solid-phase carriers and prepare enzyme conjugates to establish this method. If the source of the antibody is antiserum, the antibodies used for coating and enzyme labeling are best taken from different species of animals. If monoclonal antibodies are used, two monoclonal antibodies directed against different determinants on the antigen are generally selected for coating the solid-phase carrier and preparing enzyme conjugates respectively. This two-site sandwich method has high specificity, and can heat the test specimen and enzyme-labeled antibody together for one-step detection.

In the one-step assay, when the content of the tested antigen in the specimen is high, the excess antigen binds to the solid-phase antibody and the enzyme-labeled antibody, and no longer forms a “sandwich complex”. Similar to the post-banding phenomenon of antigen excess in the precipitation reaction, the absorbance value of the color developed after the reaction (located on the excess antigen zone) is the same as the absorbance value of a certain antigen concentration in the standard curve (located on the excess antibody zone). Normally, the result will be lower than the actual content. This phenomenon is called hook effect, because the standard curve bends like a hook after reaching the peak. When the hook effect is severe, the reaction may not show color and false negative results may appear. Therefore, when using one-step reagents to determine substances that can be abnormally increased in samples (such as serum HBsAg, AFP, and urine hCG, etc.), attention should be paid to the highest value of the measurable range. The preparation of such reagents with high-affinity monoclonal antibodies can weaken the hook effect.

If there are multiple identical determinants at different sites of the tested molecule, for example, the a determinant of HBsAg, the same monoclonal antibody for this determination can also be used to coat the solid phase and prepare the enzyme conjugate respectively. However, attention should be paid to the subtype problem in the detection of HBsAg. HBsAg has four subtypes: adr, adw, ayr, and ayw. Obviously, each subtype has the same reactivity of a determinant. This is also the use of monoclonal antibodies as a sandwich method. Attention issues.

Another note of the double antibody sandwich method for antigen detection is the interference of rheumatoid factor (RF). RF is an autoantibody, mostly of IgM type, which can bind to the Fc segment of a variety of animal IgG. If the serum specimen used for double-antibody sandwich method detection contains RF, it can act as an antigen component and bind to the solid-phase antibody and enzyme-labeled antibody at the same time, showing a false positive reaction. The use of F (ab’) or Fab fragments as reagents for enzyme conjugates can eliminate RF interference due to the removal of the Fc section. Whether the double-antibody sandwich ELISA reagent is affected by RF has been listed as an evaluation indicator for this type of reagent.

The double antibody sandwich method is suitable for the determination of bivalent or higher molecular antigens, but it is not suitable for the determination of haptens and small molecule monovalent antigens, because it cannot form a two-point sandwich.

(2) Double antigen sandwich method for antibody detection

The reaction mode is similar to the double antibody sandwich method. Coating with specific antigens and preparing enzyme conjugates to detect the corresponding antibodies. The difference from the indirect method of antibody detection is that enzyme-labeled antigen is used instead of enzyme-labeled anti-antibody. In this method, the tested specimen does not need to be diluted and can be directly used for determination, so its sensitivity is relatively higher than that of the indirect method. This method is often used for the detection of anti-HBs in hepatitis B markers. The key to this method is the preparation of enzyme-labeled antigens, and a suitable labeling method should be found according to the structure of the antigen.

(3) Indirect method for antibody detection

The indirect method is a commonly used method for detecting antibodies. The principle is to use enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect the test antibody bound to the solid-phase antigen, so it is called the indirect method (see Figure 2-3). The operation steps are as follows:

(1) Link the specific antigen with the solid-phase carrier to form a solid-phase antigen. Wash to remove unbound antigen and impurities.

(2) Add diluted test serum to keep the reaction warm. The specific antibody in the serum binds to the solid-phase antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies are left on the solid-phase carrier, and other components in the serum are washed away during the washing process.

(3) Add enzyme-labeled anti-antibody. Enzyme-labeled anti-human Ig can be used to detect total antibodies, but enzyme-labeled anti-human IgG is generally used to detect IgG antibodies. The antibody in the solid-phase immune complex binds to the enzyme-labeled antibody, thereby indirectly labeling the enzyme. After washing, the amount of enzyme on the solid-phase carrier is positively correlated with the amount of antibody tested in the specimen.

(4) Add substrate to develop color

This method is mainly used for the detection of pathogen antibodies for the diagnosis of infectious diseases. The advantage of the indirect method is that as long as the coating antigen is changed, the same enzyme-labeled anti-antibody can be used to establish a method for detecting the corresponding antibody.

The key to the success of the indirect method is the purity of the antigen. Although sometimes coating with crude antigen can achieve practical results, it should be purified as much as possible to improve the specificity of the test. Special attention should be paid to removing impurities that can react with the serum of ordinary healthy people. For example, recombinant antigens with E.Coli as engineered enzymes, if they contain E.Coli ingredients, are likely to be anti-E.Coli in the serum of people who have been infected with E.Coli. Coli antibody reacts. Antigens should not contain substances that react with enzyme-labeled anti-human Ig, such as antigens from human plasma or human tissues. If the Ig is not removed, false positive reactions will occur in the test. In addition, if the antigen contains irrelevant proteins, it will also affect the coating effect due to competitive adsorption.

Another interference factor in the indirect method is the high concentration of non-specific antibodies contained in normal serum. The specific IgG tested in the patient’s serum accounts for only a small part of the total IgG. IgG has strong adsorptivity, and non-specific IgG can be directly adsorbed to the solid-phase carrier, and sometimes can be adsorbed to the surface of the coated antigen. Therefore, in the indirect method, the antigen is usually coated with an unrelated protein (such as bovine serum albumin) again to block the empty space on the solid phase. In addition, the specimen must be diluted (1:40~1:200) in the testing process to avoid excessive negative background from affecting the judgment of the results.

 (4) Antibody test by competition method

When the interfering substance in the antigen material is not easy to remove, or it is difficult to obtain sufficient purified antigen, this method can be used to detect specific antibodies. The principle is that the antibody in the specimen competes with a certain amount of enzyme-labeled antibody to bind 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 is lighter than the negative reaction. If the antigen is of high purity, it can be directly coated on the solid phase. If there are interfering substances in the antigen and direct coating is not easy to succeed, the capture coating method can be used, that is, the antibody corresponding to the solid-phase antigen is first coated, and then the antigen is added to form the solid-phase antigen. Wash to remove impurities in the antigen, and then add the sample and enzyme-labeled antibody to compete for binding reaction. The competition method has many modes for antibody detection. The specimen and enzyme-labeled antibody can compete with the solid-phase antigen for binding. Anti-HBc ELISA generally adopts this method. Another mode is to add the sample and antigen to the solid phase antibody for competitive binding, and then add the enzyme-labeled antibody after washing to react with the antigen bound to the solid phase. This method is generally used for detection of anti-HBe.

(5) Test antigen by competition method

The small molecule antigen or semi-antibody lacks more than two sites that can be used as the sandwich method. Therefore, the double antibody sandwich method cannot be used for the determination, and the competition method can be used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen to bind to the solid-phase antibody. The more the amount of antigen in the specimen, the less the enzyme-labeled antigen bound to the solid phase, and the lighter the final color. This method is often used for ELISA determination of small molecule hormones and drugs.

(6) Antibody detection by capture coating method (classical method)

The detection of IgM antibodies is used in the early diagnosis of infectious diseases. Indirect ELISA is generally only suitable for the detection of total antibodies or IgG antibodies. For example, the indirect method of antigen-coating is used to directly measure IgM antibodies, because there are generally higher concentrations of IgG antibodies in the specimen at the same time, the latter will compete to bind to the solid phase antigen and some of the IgM antibodies cannot bind to the solid phase. Therefore, if anti-human IgM is used as a secondary antibody to determine IgM antibodies indirectly, the specimen must be treated with protein A or anti-IgG antibody to remove the interference of IgG. The capture coating method is often used in the determination of antibody IgM in clinical tests. The solid phase is first coated with anti-human IgM antibody to capture IgM in the serum sample (including specific IgM antibody against antigen and non-specific IgM). Then add the antigen, this antigen only binds to specific IgM. Then add enzyme-labeled specific antibodies against the antigen. Then it interacts with the substrate, and the color is positively correlated with the IgM in the specimen. This method is often used for the early diagnosis of viral infections. The detection mode of hepatitis A virus (HAV) antibody is shown in Figure 2-7. Rheumatoid factor (RF) can also interfere with the detection of IgM antibodies by the capture coating method, resulting in false positive reactions. Therefore, the indirect method of neutralizing IgG has recently been favored, and the detection of anti-CMV IgGM and anti-Toxoplasma IgM antibodies with such reagents has been successful.

 (7) ABS-ELISA method

ABS is an abbreviation for avidin and biotin system. Avidin is a glycoprotein with a molecular weight of 60,000. Each molecule consists of 4 subunits that can bind to biotin. Biotin is a small molecule compound with a molecular weight of 244. Derivatives made by chemical methods-hydroxysuccinimide esters can form biotin labeled products with various types of large and small molecules such as proteins and sugars. The labeling method is quite simple. The combination of biotin and avidin has a strong specificity, and its affinity is much greater than that of the antigen-antibody reaction. Once the two are combined, they are extremely stable. Since one avidin can bind to four biotin molecules, the ABS and ELISA methods can be divided into enzyme-labeled avidin-biotin (LAB) method and bridging avidin-biotin (ABC) method. Kind of type. Both use biotin-labeled antibodies (or antigens) instead of enzyme-labeled antibodies (antigens) in the original ELISA system. In LAB, solid-phase biotin is first reacted with unlabeled avidin, and then enzyme-labeled biotin is added to further increase sensitivity. In the early days, avidin was extracted from egg white, which is a basic glycoprotein and has a strong adsorption to polystyrene carrier. It can increase the background when used in ELISA. Streptavidin extracted from Streptomyces has no such shortcomings and has a tendency to replace the former in ELISA applications. Because ABS-ELISA uses two more reagents than ordinary ELISA, the operation steps are added, and ABS-ELISA is not used much in clinical testing. This method is often used in scientific research projects to detect trace components such as cytokines.

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