introduction
In 1971, Engvall et al. in Sweden used cellulose and polystyrene tubes as solid phase carriers to adsorb antigens/antibodies, and established Enzyme Linked Immunosrbent Assay (ELISA). Later, people switched to plate-type reaction wells. ELISA greatly increased the experimental throughput. The ELISA experiment is extremely flexible. In practice, people can perform various kinds of ELISA for reaction determination according to their own needs and flexible combination of materials on hand. In addition, related reagents and consumables in ELISA experiments. Both have been commercialized, so ELISA is widely used in various studies of biology. It can be seen from the ScienceDirect search that in 1975 only one document was researched for ELISA, and in 1976 it was six, but by 2010 this figure had risen to 1994, and the literature has accumulated 29,490 articles over the years, which shows that it has a wide range of research. 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 (measured antibody or antigen) 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.
Types and principles of ELISA
There are different opinions on the classification of ELISA, and different literatures have different classification opinions on principles or operations. Here, the ELISA is divided into the following four categories, and then detailed for each category: (1) direct ELISA; (2) indirect ELISA; (3) sandwich ELISA; (4) competitive inhibition ELISA. All other ELISAs are affiliated with or derived from these four types of ELISA combinations.
[direct ELISA]
Direct ELISA is the simplest step in all ELISAs. The method is to dilute the antigen in a certain proportion with a coating buffer and coat it onto the solid phase carrier. After the coating is completed, simply wash it, then add the blocking solution and block. After the end, the excess blocking solution was washed again, and the diluted specific enzyme-labeled antibody was added thereto. After incubation at 37 ° C for one hour or 4 ° C overnight, the excess antibody was removed by washing, the substrate was added for color development and the result was interpreted. The depth of the final color development is directly proportional to the amount of enzyme-labeled antibody added. The principle of direct ELISA is shown in Figure 1:
Figure 1 Direct ELISA principle
A very common example of direct ELISA is to detect the monoclonal antibody subtype. In practice, the affinity-purified mAb can be coated on the microtiter plate, and after blocking, the enzyme-labeled secondary antibody is added, and finally the substrate is colored. Just fine. In addition, direct ELISA can be used to detect serum species, and direct ELISA is used for preliminary screening in monoclonal antibody preparation. However, although the direct ELISA procedure is simple and the steps are relatively concise, its application range is very limited. An important reason is that this ELISA only undergoes one-step signal amplification (enzyme amplification), so its sensitivity is not very high. In addition, the objects to be measured are also very limited, and only the molecules labeled with the enzyme can be determined.
ã€Indirect ELISA 】
The step of the indirect ELISA is basically the same as the previous part of the direct ELISA step, except that the indirect ELISA binds to the coated antigen without the enzyme-labeled antibody, but is not the enzyme-labeled, and additionally introduces the second antibody (ie, two Anti-), the secondary antibody is enzymatically labeled, and it specifically binds to the first antibody (an antibody that binds directly to the antigen, ie, the primary antibody). Finally add the substrate to develop color and interpret the results. When the concentration of the secondary antibody is constant, the final color development result is positively correlated with the amount of primary antibody. The operation of the indirect ELISA is shown in Figure 2. The antigen is coated onto the microplate with the direct ELISA, washed and blocked, and after washing again, the diluted antibody to be tested (primary antibody) is added, and after incubation, the antigen is not washed. The primary antibody is then added to the enzyme-labeled secondary antibody and incubated again. At this time, the enzyme-labeled secondary antibody can be combined with the primary antibody, and finally the substrate is added for color development.
Since the secondary antibody is generally polyclonal, a primary antibody can bind multiple secondary antibodies, and a secondary antibody can be labeled with multiple enzyme molecules, so when the antibody to be tested is polyclonal, it can be composed of multiple The primary antibody binds to the antigen, and the signal is amplified in two steps, ultimately improving the sensitivity of the assay. In addition, since the preparation of the secondary antibody is relatively easy, and commercialization is started very early, the operator does not need to carry out the enzyme labeling of the primary antibody, which greatly reduces the workload. Indirect ELISA is a very important experimental process in the detection of antibody titer, serum titer and screening of monoclonal antibodies. In clinical diagnosis, indirect ELISA is also an important means to detect marker antibodies.
Figure 2 Principle of indirect ELISA
[Sandwich ELISA]
Sandwich ELISA can be divided into two types as a whole: direct sandwich ELISA and indirect sandwich ELISA. The direct sandwich ELISA was further divided into a double-antibody sandwich ELISA and a double antigen sandwich ELISA. The double-anti-sandwich ELISA method comprises: coating the first antibody (capture antibody) on a solid phase carrier, blocking the antigen to be detected after blocking, adding a second antibody (detection antibody), capturing antibody and detecting antibody after incubation. It may be two monoclonal antibodies against different epitopes, or one monoclonal antibody and one polyclonal antibody against the same antigen, but the detection antibody needs to be labeled with an enzyme. The principle and operation of the double antigen sandwich ELISA is basically the same as that of the double anti-sense sandwich. The difference is that the coating is antigen, the object to be tested is an antibody, and then the enzyme-labeled antigen is added, and the substrate is colored. The principle of direct sandwich ELISA is shown in Figure 3.
Figure 3 Direct sandwich ELISA principle
For double-antibody sandwich ELISA, the subject to be tested must include two or more epitopes, otherwise the detection antibody cannot bind to the antigen to be tested. For example, hapten and small molecule antigens cannot be detected by double-antibody sandwich ELISA. For the double antigen sandwich ELISA, the operation is basically the same as the indirect ELISA, but the specific antigen is used instead of the enzyme secondary antibody, so the specificity is better than the indirect method. In addition, since the secondary antibodies used in the indirect ELISA generally only recognize IgG, and any similar immunoglobulin in the double antigen sandwich ELISA can be detected, the double antigen sandwich ELISA is more sensitive than the indirect ELISA.
Indirect sandwich ELISA is a sandwich ELISA based on antibodies from two different species. The principle is to coat a specific antibody of a species source on a solid support (as a capture antibody), block, add the antigen to be tested, and warm. After breeding, add a specific antibody of a different species (non-enzyme label, as a detection antibody), and finally add an enzyme-labeled secondary antibody (specific recognition detection antibody), and then add a substrate to develop color. The principle of indirect sandwich ELISA is shown in Figure 4. Compared with the direct double-anti-sandwich ELISA, the indirect sandwich ELISA introduced an enzyme-labeled secondary antibody that specifically recognizes the detection antibody, which is equivalent to a one-step amplification system for the signal of the whole system, so the final result is more sensitive than the direct double-antibody sandwich ELISA. . At the same time, since the enzyme-labeled secondary antibody in the indirect sandwich ELISA can only recognize the detection antibody and cannot recognize the capture antibody, the specificity of the system is also guaranteed. It is worth mentioning that the indirect sandwich ELISA is not strictly required to capture the antibody from the same species as the detection antibody, or it may be the case that only the Fab fragment of the specific antibody is coated with the solid phase carrier as the capture antibody, but not The treated specific antibody of the same species is used as the detection antibody, and the enzyme-labeled secondary antibody selects the secondary antibody only for the Fc segment of the detection antibody, and such an enzyme-labeled secondary antibody also cannot recognize the capture antibody, thereby making the system The capture antibody and the detection antibody function similarly to different species sources. Indirect sandwich ELISA involves a complex system, and the reagents used are more complex than other ELISAs, but its superiority in detection sensitivity makes it widely used in the detection of samples with low abundance antigens.
Figure 4 Indirect sandwich ELISA
[competitive inhibition ELISA]
The competitive inhibition ELISA is also called blocking ELISA. The main principle is to use the antigen or antibody to be tested to interfere with the pre-designed system. The final coloration result is negatively correlated with the interference degree of the antigen or antibody to be tested. Competitive inhibition ELISA is very flexible, and based on this, a more complex experimental scheme can be designed to derive a special ELISA method such as direct competition inhibition ELISA, indirect competition inhibition ELISA, sandwich competition ELISA. Here, the principle is briefly explained by taking the direct competition inhibition ELISA and the indirect competition inhibition ELISA as examples. In the direct competition inhibition ELISA, the antigen is previously coated on a solid phase carrier, and an enzyme-labeled specific antibody is added. In the experiment, the antigen (or antibody) to be tested is added, and if the object to be tested is an antigen, the antigen to be tested competes with the antigen pre-coated on the solid phase carrier for the enzyme-labeled antibody; if the object to be detected is an antibody, the antibody is to be detected. The test antibody competes with the original enzyme-labeled antibody in the system to bind the antigen coated on the solid support. The washing process can wash away the enzyme-labeled antibody under competition, and finally add the substrate to develop color. The result of the final color development is inversely proportional to the amount of the original (or antibody) to be tested. The principle of direct competition inhibition ELISA is shown in Figure 5. Note that in the direct competition inhibition ELISA, the same pre-prepared system can measure both the antigen and the antibody.
Figure 5 Principle of direct competitive inhibition ELISA
Like the indirect ELISA, the direct competitive inhibition ELISA also has a two-step signal amplification process, so its sensitivity is also relatively high, but after pre-coating the solid phase carrier and adding the enzyme-labeled antibody, the experiment only needs to dilute the antigen or antibody to be tested. It can be added to the system for reaction, which greatly simplifies the operation of the ELISA. Kits using this system such as Germany (LDN) cortisol, DHEA-S, Estradiol and the like are used.
An indirect competitive inhibition ELISA can be viewed as interfering with a previously prepared indirect ELISA system with the antigen to be tested or the antibody to be detected. The specific principle is that the antigen is coated on a solid phase carrier, and a specific antibody and an enzyme-labeled secondary antibody corresponding to the antibody are sequentially added as a pre-prepared system. In the experiment, the diluted antigen (or antibody) to be tested is added, and the antigen (or antibody) in the sample to be tested competes with the antigen (or antibody) bound to the solid phase carrier in the pre-prepared system to bind to the specific antibody (or The antigen bound to the solid support). As with indirect ELISA, the indirect competitive inhibition ELISA also has a three-step signal amplification with a higher sensitivity than the direct competitive inhibition ELISA. The kit (PHOENIX) ACTH, which is used in the CRF indicator.
It can be seen from the above principle that competitive inhibition ELISA requires very little reagent, and both monoclonal antibody and polyclonal antibody can be used for experiments, and more importantly, whether the subject is a macromolecular substance or a polypeptide, a small molecule drug, or small A competitive inhibition ELISA can be used for molecules such as molecular hormones that have only one epitope that cannot be used in a sandwich ELISA. In addition, in the detection of hepatitis B markers, e antigen is very unstable and easily degraded into a core antigen. Therefore, sandwich ELISA cannot be used to detect e antibodies, and only competitive inhibition ELISA can be used.
In addition to the basic ELISAs described above, one can also flexibly design other types of ELISAs according to their actual needs, such as the introduction of non-enzyme-labeled secondary antibodies or ProteinA, ProteinG, etc. to increase the loading of solid-phase carriers or increase the specificity of the system. Sex and so on.
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