1. Basic Principles

In 1971, Engvall and Perlmann published an article on enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of IgG, which made the enzyme-labeled antibody technology for antigen localization developed in 1966 to be used for the determination of trace substances in liquid samples.

The basic principle of this method is:

1. The antigen or antibody is bound to the surface of a certain solid phase carrier, and its immune activity is maintained.
2. The antigen or antibody is linked with 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 measurement, the test sample (the antibody or antigen in it) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid phase carrier according to different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by the washing method, and the amount of enzyme bound to the solid-phase carrier is proportional to the amount of the tested substance in the specimen. After adding the substrate of the enzyme 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 tested substance in the sample, so qualitative or quantitative analysis can be carried out according to the depth of the color reaction. Due to the high catalytic efficiency of the enzyme, the reaction effect can be greatly amplified, so that the assay method can achieve high sensitivity.

2. Method Types and Operation Steps

ELISA can be used to measure antigens as well as antibodies. There are 3 necessary reagents in this assay:

①Solid-phase antigen or antibody;

②Enzyme-labeled antigen or antibody;

③ Substrate for enzyme action;

Various types of detection methods can be designed according to the source of the reagents, the properties of the specimen and the conditions for detection.

(1) Avidin biotin ELISA

This method is currently the most commonly used method by domestic mainstream ELISA kit manufacturers. Some manufacturers call it the double-antibody sandwich method, but it is very different from the real "double-antibody sandwich method".

Avidin is a glycoprotein that can be extracted from egg whites. The molecular weight is 60kD, and each molecule is composed of 4 subunits, which can be closely combined with 4 biotin molecules. Now more is used from streptavidin (strepavidin) extracted from Streptomyces. Biotin, also known as vitamin H, has a molecular weight of 244.31 and exists in egg yolks. The derivatives made by chemical methods, biotin-hydroxysuccinimide (BNHS), can form biotinylated products with various types of large and small molecules such as proteins, carbohydrates and enzymes. Although the combination of avidin and biotin is not an immune reaction, it has strong specificity and high affinity, and once the two are combined, they are extremely stable. Since one avidin molecule has four binding sites for biotin molecules, more biotinylated molecules can be linked to form a lattice-like complex. Therefore, coupling avidin and biotin to ELISA can greatly improve the signal intensity of ELISA.

The application of the avidin-biotin system in ELISA has various forms, which can be used for indirect coating and can also be used for final reaction amplification. Avidin can be pre-coated on the solid phase. The antibody or antigen that was originally coated on the solid phase by adsorption method is combined with biotin, and the biotinylated antibody or antigen is solidified by the avidin-biotin reaction. . This coating method can not only increase the amount of adsorbed antibody or antigen, but also fully expose the binding site. In addition, the enzyme-labeled antibody in conventional ELISA can also be replaced by a biotinylated antibody, and then an avidin-enzyme conjugate is attached to amplify the reaction signal.

Figure 1 shows the process of the ABC-ELISA (avidin biotin complex-ELISA) sandwich method for antigen detection.

(2) Competitive method

The competition method (Figure 2-1) can be used to measure antigens as well as antibodies. Taking the test antigen as an example, the test antigen and the enzyme-labeled antigen compete for binding with the solid phase antibody, so the amount of the enzyme-labeled antigen bound to the solid phase is inversely proportional to the amount of the test antigen. The operation steps are as follows:

①Connect the specific antibody to the solid-phase carrier to form a solid-phase antibody. washing.

②Add the mixed solution of the test sample and a certain amount of enzyme-labeled antigen to the test tube to react with the solid-phase antibody. If there is no antigen in the tested sample, the enzyme-labeled antigen can be successfully combined with the solid phase antibody. If the tested sample contains antigen, the enzyme-labeled antigen will bind to the solid-phase antibody at the same opportunity as the enzyme-labeled antigen, which competitively takes up the opportunity for the enzyme-labeled antigen to bind to the solid-phase carrier. amount decreased. Only the enzyme-labeled antigen is added to the reference tube. After incubation, the binding of the enzyme-labeled antigen to the solid-phase antibody can reach the most sufficient amount. washing.

③Color development by adding substrate: The color of the reference tube is the darkest due to the most bound enzyme-labeled antigen. The difference between the color depth of the reference tube and the color depth of the test tube represents the amount of antigen in the tested sample. The lighter the color of the tube to be tested, the more antigen content in the sample.

Figure 2 Schematic diagram of antigen detection by Competitive method

(3) Double antibody sandwich method

Some manufacturers habitually call the products made by the double-antibody sandwich method "one-step method", "reduced step method", "one step", "quick" and other various names, but the real operation steps and two-site one-step method are different. There is a big difference in operation.

The double-antibody sandwich method

(Figure 3) is a commonly used method for antigen detection. The operation steps are as follows:

①Connect the specific antibody to the solid-phase carrier to form a solid-phase antibody: wash to remove unbound antibodies and impurities.

②Add the test sample: make it contact and react with the solid-phase antibody for a period of time, and let the antigen in the sample combine with the antibody on the solid-phase carrier to form a solid-phase antigen complex. Washing removes other unbound material.

③Add enzyme-labeled antibody: combine the antigen on the solid-phase immune complex with the enzyme-labeled antibody. Thoroughly wash unbound enzyme-labeled antibody. At this time, the amount of the enzyme carried on the solid-phase carrier is positively correlated with the amount of the tested substance in the sample.

④Substrate addition: The enzyme in the sandwich complex catalyzes the substrate to become a colored product. The characterization or quantification of the antigen is carried out according to the degree of color reaction.

According to the same principle, the macromolecular antigens are prepared as solid-phase antigens and enzyme-labeled antigen conjugates, respectively, and the double-antigen sandwich method can be used to measure the antibodies in the specimen.

(4) Two-site one-step method

When the double antibody sandwich method is used to determine the antigen, if the monoclonal antibodies against two different antigenic determinants on the antigen molecule are used as the solid-phase antibody and the enzyme-labeled antibody, respectively, the addition of the sample and the enzyme-labeled antibody can be used in the determination. Two steps and one step (Figure 2). This two-site step not only simplifies the operation, shortens the reaction time, such as the application of high-affinity monoclonal antibodies, the sensitivity and specificity of the assay are also significantly improved. The use of monoclonal antibodies has brought the ELISA for antigen determination to a new level.

Figure 4. Schematic diagram of two-site one-step method

In the one-step assay, attention should be paid to the hook effect, which is similar to the post-band phenomenon of excess antigen in the precipitation reaction. When the concentration of the antigen to be tested in the sample is quite high, the excess antigen is combined with the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a sandwich complex, and the result will be lower than the actual content. False-negative results can even occur when the hook effect is severe.

(5) Indirect detection of antibodies

The indirect method (Figure 5) is the most commonly used method for detecting antibodies. The operation steps are as follows:

①Connect the specific antigen to the solid-phase carrier to form a solid-phase antigen: wash to remove unbound antigen and impurities.

②Add the diluted test serum: the specific antibody in it is combined with the antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies remain on the solid phase support. Impurities in other immunoglobulins and serum are washed away during the washing process because they cannot bind to the solid-phase antigen.

③Enzyme-labeled anti-antibody: binds 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, to measure human antibodies to a certain disease, enzyme-labeled goat anti-human IgG antibodies can be used.

④Color development by adding substrate: the color depth represents the amount of the tested antibody in the sample.

In this method, as long as different solid-phase antigens are replaced, the same enzyme-labeled antibody can be used to detect various target antibodies corresponding to the antigens.

Figure 5. Schematic diagram of indirect method for antibody detection

(6) Detection of IgM antibodies by capture method

Capture coating method (also known as reverse indirect method) ELISA is mainly used for the determination of certain antibody subtype components (such as IgM) in serum. Taking the currently commonly used IgM assay as an example, due to the presence of specific IgM and IgG against a certain antigen in serum, IgG can interfere with the determination of IgM. Therefore, all serum IgM (including heterozygous IgM and non-specific IgM) are first immobilized on the solid phase, and then specific IgM is determined after removing IgG. The detection of IgM antibodies is used in the early diagnosis of epidemic diseases. The solid phase is first coated with anti-human IgM antibodies to capture IgM in serum samples (both antigen-specific and non-specific IgM). Antigen is then added, which binds only to specific IgM. Enzyme-labeled specific antibodies against the antigen are then added. When it interacts with the substrate, the coloration is positively correlated with the IgM in the specimen. This method is often used for early diagnosis of viral infections. Rheumatoid factor (RF) can also interfere with the capture-coating assay for IgM antibodies, resulting in false positive responses. Therefore, indirect methods of neutralizing IgG have recently become popular, and the detection of anti-CMV IgGM and anti-Toxoplasma IgM antibodies with these reagents has been successful. The operation steps are as follows:

Figure 6. Schematic diagram of IgM antibody measurement by capture method

①The anti-human IgM antibody is linked to a solid phase carrier to form a solid phase anti-human IgM. washing.

②Add diluted serum samples: After the incubation reaction, the IgM antibody in the serum is captured by the solid phase antibody. Washing removes other immunoglobulins and impurities in serum.

③Add specific antigen reagent: it only binds to specific IgM on the solid phase. washing.

④Add specific enzyme-labeled antibody: make it react with the antigen bound on the solid phase. washing.

⑤Color development with substrate: if color is displayed, it means that the specific IgM antibody in the serum sample exists, which is a positive reaction.

Technical points of ELISA

The technical points of ELISA include three aspects: preparation of reagents, selection of reaction conditions and standardization of operation.

Preparation of reagents

The main reagents of ELISA are solid-phase antigens or antibodies, enzyme-labeled antigens or antibodies, and enzyme reaction substrates directly associated with the labeled enzymes. The raw materials and preparation methods of these reagents are described below.

Solid phase carrier

There are many substances that can be used as carriers in ELISA, the most commonly used is polystyrene. Polystyrene has strong protein adsorption properties, and the original immune activity is retained after the antibody or protein antigen is adsorbed on it. Polystyrene is a plastic that can be made in various forms. During the ELISA assay, it acts as a carrier and container and does not participate in chemical reactions. Coupled with its low price, it is widely used. There are three main shapes of ELISA carriers: small test tubes, beads and micro-reaction plates. The characteristic of the small test tube is that it can also be used as a reaction container, and finally put into the spectrophotometer for colorimetry. The beads are generally spheres with a diameter of 0.6 cm, and the adsorption area is greatly increased after the surface is frosted. If a special washer is used, the balls are rolled and rinsed during the washing process, and the effect is better. The most commonly used carrier is the micro-reaction plate, and the product specially used for ELISA assay is also called ELISA plate. The international standard plate shape is 8×12 96-well format. In order to facilitate the detection of a small number of specimens, there are 8- or 12-well strips, which are the same size as standard ELISA plates after being placed in the stand. The ELISA plate is characterized by the simultaneous detection of a large number of samples and the rapid readout of the results on a specific colorimeter. Now there are a variety of automated instruments for ELISA detection of micro-reaction plate type, including steps such as sample addition, washing, incubation, and colorimetry, which are extremely beneficial to the standardization of operations. A good ELISA plate should have good adsorption performance, low blank value, high transparency at the bottom of the well, and similar performance between plates and between wells in the same plate. Due to the difference in ingredients and the difference in the production process of polystyrene ELISA plates, the quality of various products varies greatly. Therefore, each batch of polystyrene products must be checked for performance before use. The commonly used inspection method is: after coating each well of ELISA plate with a certain concentration of human IgG (usually 10ng/ml), add appropriately diluted enzyme-labeled anti-human IgG anti-human IgG antibody to each well, wash after incubation, and add bottom. After the enzyme reaction was terminated, the absorbance of each well solution was measured. The reaction conditions were controlled so that each reading was around 0.8. Calculate the average of all readings. All individual readings should differ by less than 10% from the average reading. A plastic similar to polystyrene is polyvinyl chloride. As an ELISA solid phase carrier, polyvinyl chloride plate is characterized by thin soft plate, can be cut, cheap, but the finish is not as good as polystyrene plate. The adsorption performance of polyvinyl chloride for protein is higher than that of polystyrene, but the blank value is sometimes slightly higher. In order to compare the advantages and disadvantages of different solid phases in an ELISA assay, the following methods can be used to test: select a typical positive sample and a typical negative sample with other immunological assay methods. They were respectively diluted in a series, measured on different solid-phase supports according to the predetermined ELISA procedure, and then the measured results were compared. On which carrier the positive result and the negative result can be most distinguished, this carrier is the most suitable solid phase carrier for this ELISA assay. In addition to plastic products, there are two materials for the carrier of solid-phase enzyme immunoassay: one is microporous membrane, such as nitrocellulose membrane, nylon membrane, etc. This type of assay will be described in "Enzyme Immunoassay of Membrane Carrier" introduce. The other carrier is made of iron-containing magnetic particles. During the reaction, the solid-phase particles are suspended in the solution and have the rate of liquid-phase reaction. After the reaction, magnet attraction is used as a means of separation, and washing is also very convenient, but it needs to be equipped with special instruments.