What are the ELISA methods?

Enzyme linked immunosorbent assay (Enzyme Linked ImmunoSorbent Assay, ELISA) is an important method in the study of antibody. It uses the specific binding of antigen and antibody to directly or indirectly bind the test substance to the enzyme-labeled antibody, and then qualitatively and quantitatively analyzes the color reaction of the labeled enzyme with the substrate. So, is it related to the sensitivity of other detection methods?

ELISA is usually divided into four types: direct method, indirect method, competition method, sandwich method and the like.

Direct method (Direct ELISA) requires only one anti-antigen and enzyme, easy to operate, can avoid cross-reactions, however, the enzyme-labeled antibody method requires high specificity requirements, but not all of a suitable anti-marking process. The direct method is rare in practical use. It does not allow quantitative analysis of antibodies in the sample. Because the antibody in the sample is a non-enzyme-labeled antibody, subsequent color development cannot be performed. However, the method is improved by competitive ELISA. Text.

Indirect method (Indirect ELISA) was used to increase the signal strength of the secondary antibody, the antibody also varied, however, prone to cross-reaction of the indirect method. The indirect method can only be used to determine antibodies, and is mainly used for the diagnosis of diseases. The advantage is that only the coated antigen needs to be changed, and the enzyme-labeled antibody is universal.

Sandwich (Sandwich ELISA) into double antibody sandwich method and the double antigen sandwich method:

In the double antibody sandwich method, the antigen is bound to different sites by two antibodies, a capture antibody and a detection antibody. The capture antibody is immobilized on a vector, and the detection antibody is subjected to color development analysis by binding to the antigen. The antibody used in the double antibody sandwich assay needs to be a monoclonal antibody and has different binding sites for the same antigen, so as to avoid cross-reaction or competitive binding of the two antibodies to the same site. The sandwich method has the advantages of high sensitivity and high specificity, but the method is only applicable to antigen detection with multiple binding sites, and is mainly used for detecting various macromolecular antigens - for example, measuring HBsAg, HBeAg in medical tests, AFP, etc. Due to the high specificity of the double antibody sandwich method, the sample and the enzyme-labeled antibody are sometimes added to the reaction during the detection process (one-step method). At this time, if the antigen content in the sample is too high, it will cause the solid phase antibody and the enzyme-labeled antibody. Both have a combination without forming a "sandwich complex", and the results measured at this time will be lower than the actual content or even the negative result (hook effect). Therefore, if the one-step method is used to determine the antigenic content of the sample, attention should be paid to the linear range of measurement, and the use of high-affinity monoclonal antibodies can also attenuate the hook effect.

In the double antigen sandwich method, the antigen is coated on the solid phase, and after the antibody in the test sample is bound to the antigen, the binding and subsequent reaction using the enzyme-labeled antigen can be used to determine the antibody in the sample. The key to the double antigen sandwich method is the preparation of the enzyme-labeled antigen, which needs to be designed according to the antigen structure. It is the method used to determine the anti-HBsAg antibody in medical tests. The sample to be tested can be directly measured without dilution, so the sensitivity is determined. Relatively higher than the indirect method.

Competition (Competitive ELISA) ELISA analysis of antibody is free antibody / antigen solid phase sample / antigen competitive binding. When the concentration of free antibody (or antigen) is higher, the less the enzyme-labeled antibody (or antigen) that can bind to the immobilized antigen (or antibody), the lighter the subsequent color development, ie, the lighter the color is compared with the control. , indicating that the antibody (or antigen) content in the test sample is higher. This method is suitable for relatively impure samples, and the repeatability is good, but the sensitivity and specificity of the detection are poor. Competitive assay antibodies are often used to detect samples that are difficult to remove with certain interfering substances and that are difficult to purify. Competitive assay antigens are commonly used to detect small molecule antigens and haptens. These antigens usually lack more than two recognition sites and are not suitable for double-antibody sandwich assays.

The capture coating method, also known as the reverse indirect method, is mainly used to determine a certain type of antibody subtype such as IgM in serum. In order to exclude the interference of IgG in serum, it is used to capture IgM in the sample after coating with anti-IgM antibody, and then add specific binding antigen for subsequent detection. Capture coating is commonly used for early diagnosis of viral infections. In the assay for the determination of IgM, interference with rheumatoid factor (RF, a self-IgM antibody that binds to various animal IgG Fc) is often encountered, resulting in a false positive response. The use of F(ab') or Fab fragments to prepare enzyme-labeled antibodies can eliminate RF interference, and this method has the same effect in the double antibody sandwich method.

In addition to these five detection methods, new ELISA is constantly being developed, for example, cell-based ELISA (cell-based ELISA), is to replace the original cells were seeded in microplates antigen, which can cause some processing The real-time dynamic protein content change is measured without lysis of cells, which can reduce the loss of the target protein, and the disadvantage is that the antigen cannot be quantitatively analyzed.