1、 The principle of ELISA

The basis of ELISA is solid-phase preparation of antigens or antibodies and enzyme labeling of antigens or antibodies. The antigen or antibody bound to the surface of the solid-phase carrier still maintains its immunological activity, while the enzyme labeled antigen or antibody retains both its immunological activity and enzyme activity. During the measurement, the tested specimen (the antibody or antigen being measured) reacts with the antigen or antibody on the surface of the solid-phase carrier. Separate the antigen antibody complex formed on the solid-phase carrier from other substances in the liquid by washing. Adding enzyme labeled antigens or antibodies also binds to the solid-phase carrier through reaction. At this point, the amount of enzyme on the solid phase is proportional to the amount of the test substance in the specimen. After adding the substrate to the enzyme reaction, 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 analyte in the specimen. Therefore, qualitative or quantitative analysis can be performed based on the depth of the color. Due to the high catalytic efficiency of enzymes, they indirectly amplify the results of immune reactions, making the measurement method highly sensitive.

2、 Types of ELISA

ELISA can be used to detect antigens as well as antibodies. In this measurement method, there are three necessary reagents: (1) solid-phase antimicrobial agents or antibodies, namely 'immunoadsorbents'; (2) Enzyme labeled antigens or antibodies are called 'conjugates'; (3) The substrate of enzyme reaction. Various types of detection methods can be designed based on the source of reagents, the condition of specimens, and the specific conditions of detection. There are several types of ELISA used for clinical testing, including:

1. Double antibody sandwich method for antigen detection

The double antibody sandwich method is a commonly used method for detecting antigens, and the operating steps are as follows:

1) Connect specific antibodies with solid-phase carriers to form solid-phase antibodies. Wash to remove unbound antibodies and impurities.

2) Add the test specimen and conduct a thermal insulation reaction. The antigen in the specimen binds to the solid-phase antibody to form a solid-phase antigen antibody complex. Wash to remove other unbound substances.

3) Add enzyme-linked antibodies and incubate the reaction. The antigen on the solid-phase immune complex binds to the enzyme-linked antibody. *Wash unbound enzyme-linked antibodies. At this point, the amount of enzyme carried on the solid-phase carrier is correlated with the amount of antigen being tested in the specimen.

4) Add substrate for color development. Enzymes on the solid phase catalyze substrates into colored products. Measure the amount of antigen in the specimen through colorimetry.

In clinical testing, this method is suitable for detecting various large molecular antigens such as HBsAg, HBeAg, AFP, hCG, etc. As long as specific antibodies against the tested antigen are obtained, this method can be established for coating solid-phase carriers and preparing enzyme conjugates. If the source of the antibody is anti serum, the antibodies used for coating and enzyme-linked immunosorbent assay are taken from different species of animals. If monoclonal antibodies are applied, two monoclonal antibodies targeting different determinants on the antigen are generally selected, which are respectively used for coating the solid-phase carrier and preparing enzyme conjugates. This double site sandwich method has high specificity and can perform one-step detection by incubating the test sample and enzyme-linked antibody together. In one-step determination, when the content of the tested antigen in the specimen is high, excess antigen binds to solid-phase antibody and enzyme-linked antibody separately, instead of forming a 'sandwich complex'. Similar to the phenomenon of antigen excess in precipitation reactions, 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). If measured by conventional methods, the result will be lower than the actual content. This phenomenon is called the hook effect, because the standard curve bends in a hook shape after reaching its peak. When the hook effect is severe, the reaction may not even show color and may result in false negative results. Therefore, when using one-step reagents to determine substances with abnormally high levels in specimens (such as HBsAg, AFP in serum, and hCG in urine), attention should be paid to the highest value within the detectable range. The preparation of such reagents using high affinity monoclonal antibodies can weaken the hook effect. If there are multiple identical determinants at different sites of the tested molecule, such as the alpha determinant of HBsAg, the same monoclonal antibody targeting this determinant can be used to coat the solid phase and prepare enzyme conjugates separately. However, in the detection of HBsAg, attention should be paid to the issue of subtypes. HBsAg has four subtypes: ADR, ADW, AyR, and AyW. Although each subtype has the same reactivity of the A determinant, this is also a concern when using monoclonal antibodies as sandwich methods. Another point to note in the double antibody sandwich method for antigen detection is the interference of rheumatoid factor (RF). RF is an autoantibody, mostly of IgM type, that can bind to the Fc segment of various animal IgG. If the serum sample used for double antibody sandwich detection contains RF, it can act as an antigen component and bind to solid-phase antibodies and enzyme-linked antibodies, exhibiting false positive reactions. The use of F (ab ') or Fab fragments as enzyme binding reagents eliminates RF interference by removing the Fc segment. Whether the double antibody sandwich ELISA reagent is affected by RF has been listed as an assessment indicator for this type of reagent. The double antibody sandwich method is suitable for determining large molecule antigens with divalent or higher valences, but it is not suitable for determining haptens and small molecule monovalent antigens because it cannot form a two site sandwich.

2. Double antigen sandwich method for antibody detection

The reaction mode is similar to the double antibody sandwich method. Coat with specific antigens and prepare enzyme conjugates to detect corresponding antibodies. The difference from indirect antibody testing is the use of enzyme-linked antigens instead of enzyme-linked antibodies. In this method, the tested sample does not need to be diluted and can be directly used for determination, so its sensitivity is relatively higher than that of indirect methods. This method is often used to detect anti HBs in hepatitis B markers. The key to this method lies in the preparation of enzyme-linked antigens, and appropriate labeling methods should be found based on the different antigen structures.

3. Indirect method for detecting antibodies

Indirect method is a commonly used method for detecting antibodies. The principle is to use enzyme labeled anti antibodies (anti human immunoglobulin antibodies) to detect the tested antibodies that bind to solid-phase antigens, hence it is called the indirect method (see Figure 2-3). The operation steps are as follows:

1) Combine specific antigens with solid-phase carriers to form solid-phase antigens. Wash to remove unbound antigens and impurities.

2) Dilute the tested serum and incubate the reaction. Specific antibodies in serum bind to solid-phase antigens to form solid-phase antigen antibody complexes. After washing, only specific antibodies remain on the solid-phase carrier, and other components in the serum are washed away during the washing process.

3) Add enzyme labeled anti antibodies. 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-linked antibody, indirectly labeling the enzyme. After washing, the enzyme amount on the solid-phase carrier is positively correlated with the amount of the tested antibody in the specimen.

4) The substrate colorimetric assay is mainly used for the detection of pathogen antibodies and the diagnosis of infectious diseases.

The advantage of indirect method is that as long as the antigen is transformed, the same enzyme-linked immunosorbent assay can be used to establish a method for detecting the corresponding antibody. The key to the success of indirect methods lies in the purity of the antigen. Although crude antigen coating can sometimes achieve practical and effective results, purification should be carried out as much as possible to improve the specificity of the experiment. Special attention should be paid to removing impurities that can react with the serum of generally healthy individuals, such as recombinant antigens using E. Coli as an engineered enzyme. If E. Coli is present, it is likely to react with anti E. Coli antibodies in the blood of individuals infected with E. Coli. Antigens should not contain substances that react with enzyme-linked anti human Ig, such as antigens from human plasma or tissue. If the Ig is not removed, false positive reactions may occur in the test. In addition, if the antigen contains unrelated proteins, it can also affect the coating effect due to competitive adsorption. Another interfering factor in indirect methods is the high concentration of non-specific substances present in normal serum. The specific IgG detected in the patient's serum only accounts for a small portion of the total IgG. IgG has strong adsorption properties, and non-specific IgG can be directly adsorbed onto solid-phase carriers, sometimes also onto the surface of the coated antigen. Therefore, in the indirect method, after antigen coating, it is generally coated again with unrelated proteins (such as bovine serum albumin) to block the empty space on the solid phase. In addition, the specimen must be diluted (1:40~1:200) during the detection process to avoid excessive negative background affecting the judgment of the results.

4. Antibody testing under competition law

When the interfering substances in the antigen material are difficult to remove or it is not easy to obtain sufficient purified antigen, this method can be used to detect specific antibodies. The principle is that antibodies in the specimen compete with a certain amount of enzyme-linked antibodies to bind to solid-phase antigens. The more antibodies there are in the specimen, the less enzyme-linked antibodies bind to the solid phase, resulting in a lighter color for positive reactions compared to negative reactions. If the antigen is of high purity, it can be directly encapsulated in a solid phase. If there are interfering substances in the antigen, direct encapsulation is not easy to succeed. The capture encapsulation method can be used, which involves first encapsulating the antibody corresponding to the solid-phase antigen, and then adding the antigen to form the solid-phase antigen. Wash to remove impurities from the antigen, then add the sample and enzyme-linked antibody for competitive binding reaction. There are multiple modes of antibody detection using the competitive method, which can compete with solid-phase antigens to bind samples and enzyme-linked antibodies. This method is generally used for anti HBc ELISA. Another mode is to add the specimen and antigen together to the solid-phase antibody for competitive binding, wash and then add the enzyme-linked antibody to react with the antigen bound to the solid-phase. The detection of anti HBe generally adopts this method.

5. Antigen testing under competition law

Due to the lack of two or more sites that can be used as sandwich methods for small molecule antigens or semi antibodies, the double antibody sandwich method cannot be used for determination, and the competitive method mode can be used. The principle is that antigens in the specimen compete with a certain amount of enzyme-linked antigen to bind with solid-phase antibodies. The higher the antigen content in the specimen, the fewer enzyme labeled antigens bound to the solid phase, and the lighter the color after extraction. This method is commonly used for ELISA detection of small molecule hormones, drugs, etc.

6. Capture package method for antibody detection

The detection of IgM antibodies is used for early diagnosis of infectious diseases. Indirect ELISA is generally only suitable for detecting total antibodies or IgG antibodies. If the indirect method of antigen encapsulation is used to directly determine IgM antibodies, there is usually a high concentration of IgG antibodies present in the specimen, which will compete to bind to the solid-phase antigen and prevent some IgM antibodies from binding to the solid-phase. Therefore, if anti human IgM is used as a secondary antibody to indirectly determine IgM antibodies, the specimen must first be treated with A protein or anti IgG antibodies to remove interference from IgG. The capture encapsulation method is commonly used in clinical testing to determine antibody IgM. First, coat the solid phase with anti human IgM antibodies to capture IgM in the serum sample (including antigen specific IgM antibodies and non-specific IgM). Then add an antigen that only binds to specific IgM. Then add enzyme labeling to specific antibodies against the antigen. Further interaction with the substrate results in a positive correlation between the color and IgM in the specimen. This method is commonly used for early diagnosis of viral infections. The detection mode of hepatitis A virus (HAV) antibodies is shown in Figure 2-7. Rheumatoid factor (RF) can also interfere with the capture package method for detecting IgM antibodies, leading to false positive reactions. Therefore, the indirect method of neutralizing IgG has recently gained popularity, and the use of such reagents to detect anti CMV IgGM and anti Toxoplasma IgM antibodies has been successful.

7. ABS-ELISA method

ABS is an abbreviation for avidin biotin system. Affinity protein is a glycoprotein with a molecular weight of 60000, each molecule consisting of four subunits that can bind to biotin. Biotin is a small molecule compound with a molecular weight of 244. The derivative hydroxysuccinimide ester made by chemical methods 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. The binding of biotin and avidin has strong specificity, and its affinity is much greater than that of antigen antibody reactions. Once combined, the two are extremely stable. Due to the fact that one avidin can bind to four biotin molecules, ABS and ELISA methods can be divided into two types: enzyme labeled avidin biotin (LAB) method and bridged avidin biotin (ABC) method. Both use biotinylated antibodies (or antigens) instead of enzyme-linked 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 enhance sensitivity. In the early days, avidin was extracted from egg white and is an alkaline glycoprotein with strong adsorption properties to polystyrene carriers. It can be used in ELISA to increase the background. Streptomycin extracted from Streptomyces does not have this drawback and has a tendency to replace the former in ELISA applications. Due to the use of two additional reagents and increased operational steps compared to regular ELISA, ABS-ELISA is not widely used in clinical testing.