Principle of ELISA
The basis of ELISA is the solid phase of the antigen or antibody and the enzyme labeling of the 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 therein) is reacted 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. Enzyme-labeled antigens or antibodies are added, and are 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 for the enzyme reaction, the substrate is catalyzed by the enzyme into 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 performed according to the depth of the color. Due to the high catalytic efficiency of the enzyme, the results of the immune response are indirectly amplified, making the assay method highly sensitive.
2. Types of ELISA
ELISA can be used to measure antigens and antibodies. There are three necessary reagents in this measurement method: (1) solid phase probiotic or antibody, namely "immunosorbent"; (2) enzyme-labeled antigen or antibody, called "conjugate" ( conjugate); (3) Substrate for enzyme reaction. According to the source of the reagent and the condition of the specimen and the specific conditions of the test, various types of detection methods can be designed. There are several types of ELISA used for clinical testing:
2.2.1 Double antibody sandwich method for antigen detection
The double antibody sandwich method is the most commonly used method for antigen detection. The 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 specimen to be tested and keep it warm. The antigen in the specimen combines with the solid-phase antibody to form a solid-phase antigen-antibody complex. Wash to remove other unbound materials.
3) Add enzyme-labeled antibody and incubate the reaction. The antigen on the solid-phase immune complex binds to the enzyme-labeled antibody. Wash unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme carried on the solid-phase carrier is related to the amount of tested antigen in the specimen.
4) Add substrate to develop color. The enzyme on the solid phase catalyzes the substrate into a colored product. By colorimetry, measure the amount of antigen in the specimen. In clinical testing, this method is suitable for testing large protein antigens such as various proteins, such as HBsAg, HBeAg, AFP, hCG, etc. As long as the heteroantibodies against the test antigen are obtained, it can be used to coat the solid phase carrier and prepare the enzyme conjugate to establish this method. If the source of the antibody is antiserum, the antibody used for coating and enzyme labeling is preferably obtained from animals of different species. If monoclonal antibodies are used, two monoclonal antibodies against different determinants on the antigen are generally selected for coating the solid phase carrier and preparing the enzyme conjugate. This two-site sandwich method has a high specificity, and can incubate the test specimen and enzyme-labeled antibody together for one-step detection.
In the one-step measurement, when the content of the tested antigen in the specimen is high, the excess antigen is bound to the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a "sandwich complex". Similar to the phenomenon of excess banding of antigen in the precipitation reaction, the absorbance value of the color developed after the reaction (located on the antigen excess band) is the same as the absorbance value of a certain antigen concentration in the standard curve (located on the antibody excess band) (see 1.3 .2, Figure 1-4), if measured according to the usual method, the result will be lower than the actual content, this phenomenon is called the hook effect (hook effect), because the standard curve reaches the peak after a hook-shaped curve . When the hook effect is serious, the reaction may not even develop color and a false negative result may appear. Therefore, when using a one-step reagent to determine the abnormally high content of the sample (such as HBsAg, AFP in serum and hCG in urine, etc.), you should pay attention to the highest value of the measurable range. Preparation of such reagents with high affinity monoclonal antibodies can weaken the hook effect.
If the test molecule contains multiple identical determinants, such as the a determinant of HBsAg, the same monoclonal antibody for this determination can be used to coat the solid phase and prepare the enzyme conjugate. However, in the detection of HBsAg, attention should be paid to the subtype problem. HBsAg has four subtypes: adr, adw, ayr, and ayw. Although each subtype has the same a determinant reactivity, this is also the application of sandwich antibody Note the problem.
Another note of the double antibody sandwich method for antigen detection is the interference of rheumatoid factor (RF). RF is an autoantibody, mostly IgM type, which can bind to the Fc segment of various animal IgG. If the serum sample used for the detection of the double antibody sandwich method contains RF, it can serve as an antigen component and at the same time combine with the solid-phase antibody and the enzyme-labeled antibody, showing a false positive reaction. Using F (ab ') or Fab fragments as reagents for the enzyme conjugate, due to the removal of the Fc segment, RF interference is eliminated. Whether the double-antibody sandwich ELISA reagent is affected by RF has been listed as an evaluation index for such reagents (see 6.2).
The double antibody sandwich method is suitable for the determination of bivalent or more bivalent large-molecule antigens, but it is not suitable for the determination of hapten and small-molecule monovalent antigens because it cannot form a two-point sandwich.
2.2.2 Double antigen sandwich method is similar to double antibody sandwich method in measuring antibody reaction mode. Coating with specific antigens and preparation of enzyme conjugates to detect the corresponding antibodies. The difference with the indirect method is to use enzyme-labeled antigen instead of enzyme-labeled anti-antibody. In this method, the test specimen does not need to be diluted, and can be directly used for determination, 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 of this method lies in the preparation of enzyme-labeled antigen, and the appropriate labeling method should be searched according to the structure of the antigen.
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 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) The specific antigen is connected with the solid phase carrier to form a solid phase antigen. Wash to remove unbound antigen and impurities.
2) Add diluted test serum and incubate for reaction. The specific antibodies in the serum bind 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 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) Substrate coloring This method is mainly used for the detection of pathogen antibodies and the diagnosis of infectious diseases. The advantage of the indirect method is that as long as the coated 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 coating with crude antigen can sometimes achieve practical and effective results, it should be purified as much as possible to improve the specificity of the test. Special attention should be paid to the removal of impurities that can react with the serum of general healthy people, such as recombinant antigens using E.Coli as an engineering enzyme. .Coli antibody reacts. Antigens must not contain substances that react with enzyme-labeled anti-human Ig, such as antigens from human plasma or human tissue. If Ig is not removed, false positive reactions will also occur in the test. In addition, if the antigen contains irrelevant protein, it will also affect the coating effect due to competitive adsorption.
Another interference factor in the indirect method is the high concentration of non-specificity contained in normal serum. The specific IgG detected in the patient's serum accounts for only a small part of the total IgG. IgG is very adsorbable, non-specific IgG can be directly adsorbed on the solid phase carrier, and sometimes can also be adsorbed on the surface of the coated antigen. Therefore, in the indirect method, after the antigen is coated, an extraneous protein (such as bovine serum albumin) is generally coated again to block the empty gap on the solid phase. In addition, the specimen must be diluted (1: 40 ~ 1: 200) in the test process to avoid excessively high negative background affecting the judgment of the results.
2.2.4 Competitive assay for antibodies
When the interfering substances in the antigen material are not easy to remove, or it is not easy to obtain enough 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 for binding to the solid-phase antigen. The more antibody in the specimen, the less 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 with the solid phase. If there are interfering substances in the antigen, direct coating is not easy to succeed, you can use the capture coating method, that is, first coat the antibody corresponding to the solid phase antigen, and then add the antigen to form the solid phase antigen. Wash to remove impurities in the antigen, and then add specimens and enzyme-labeled antibodies for competitive binding reaction. There are multiple modes for the detection of antibodies by the competition method. The specimen and the enzyme-labeled antibody can be competitively combined with the solid-phase antigen. This method is generally used for anti-HBc ELISA. Another mode is to add the specimen and the antigen to the solid-phase antibody together for competitive binding. After washing, the enzyme-labeled antibody is added to react with the antigen bound to the solid phase. Anti-HBe detection generally uses this method.
2.2.5 The competition method for the detection of small antigens or half-antigens lacks more than two sites that can be used as a sandwich method. Therefore, the double antibody sandwich method cannot be used for measurement, and the competition method mode can be used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen for binding 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.
2.2.6 Antibody detection by capture coating method
The 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 indirect method of antigen coating is used to directly determine the IgM antibody, there is generally a higher concentration of IgG antibody in the specimen, which 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 anti-human IgM is used as the secondary antibody to indirectly determine the IgM antibody, the specimen must be treated with protein A or anti-IgG antibody to remove IgG interference. The capture coating method is often used in the determination of antibody IgM in clinical tests. The solid phase was first coated with anti-human IgM antibody to capture IgM in serum samples (including specific IgM antibodies against antigens and non-specific IgM). Then an antigen is added, which only binds to specific IgM. Enzymes are then added to label specific antibodies against the antigen. When interacting with the substrate, the color is positively correlated with the IgM in the specimen. This method is often used for 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 capture coating method to determine IgM antibodies, resulting in false positive reactions. Therefore, the indirect method of neutralizing IgG has been favored recently, and detection of anti-CMV IgGM and anti-Toxoplasma IgM antibodies with such reagents has been successful.
2.2.7 ABS-ELISA method
ABS is an abbreviation for avidin and biotin system. Avidin is a glycoprotein with a molecular weight of 60,000, and each molecule is composed of 4 subunits that can bind to biotin. Biotin is a small molecule compound with a molecular weight of 244. The derivative-hydroxysuccinimide ester prepared by chemical methods 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, its affinity is much greater than that of antigen and antibody, and the two are extremely stable once they are combined. Since one avidin can be combined with 4 biotin molecules, the ABS and ELISA methods can be divided into enzyme-labeled avidin-biotin (LAB) method and bridged avidin-biotin (ABC) method. Kind. Both use biotin-labeled antibodies (or antigens) instead of enzyme-labeled antibodies (antigens) in the original ELISA system. In LAB, solid-phase biotin first reacts with unlabeled avidin, and then enzyme-labeled biotin is added to further increase the sensitivity. In the early days, avidin was extracted from egg whites. This egg avidin is a basic glycoprotein and has strong adsorption to polystyrene carriers. It can increase the background when used in ELISA. Streptavidin extracted from Streptomyces has no such shortcomings, and it tends to replace the former in ELISA applications. Because ABS-ELISA uses two more reagents than ordinary ELISA, it increases the operation steps, and ABS-ELISA is not widely used in clinical testing.
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