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Disadvantages of Indirect ELISA

 Enzyme linked immunosorbent assay (ELISA) refers to the binding of antigen or antibody to the surface of a solid-phase carrier and maintain its immunological activity. The antigen or antibody is linked with a certain enzyme to form an enzyme-labeled antigen or antibody. This enzyme-labeled antigen or antibody retains its immune activity and enzyme activity.

Common categories of ELISA: direct ELISA, indirect ELISA, sandwich ELISA (double antibody, double antigen), competitive ELISA.

disadvantages of indirect ELISA

Disadvantages of Indirect ELISA


The indirect ELISA method is similar to the direct method. The difference is that the primary antibody is not labeled with an enzyme. Instead, an enzyme-labeled secondary antibody is used to identify the primary antibody to determine the amount of antigen. The antigen is first bound to the ELISA plate, and then the detection is performed in two steps: first, the detection antibody is added to specifically bind to the antigen, and then the enzyme-labeled secondary antibody is added for detection and the color is developed by the substrate. The indirect method is the most commonly used method for detecting antibodies. Its principle is to use enzyme-labeled antibodies to detect the test antibody that has been bound to the solid phase, so it is called the indirect method.


-The Operation Steps are as Follows:

  • ⑴ Connect the specific antigen with the solid-phase carrier to form a solid-phase antigen: washing to remove unbound antigen and impurities.
  • ⑵Add diluted test serum: the specific antibody in it combines with the antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies remain on the solid phase carrier. Other antibodies and impurities in the serum are washed away during the washing process because they cannot be combined with the solid phase antigen.
  • (3) Enzyme-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 phase carrier represents the amount of specific antibodies. For example, to test human antibodies against certain diseases, enzyme-labeled goat anti-human IgG antibodies can be used.
  • ⑷ Add substrate to develop color: the depth of color represents the amount of antibody tested in the specimen.

-Advantages of Indirect ELISA:


It is the most commonly used method for measuring Ab. The secondary antibody can enhance the signal, and there are many options for different measurement and analysis. The primary antibody without enzyme label can retain the most immunoreactivity. Compared with direct ELISA, indirect ELISA uses enzyme-labeled secondary antibody, which has higher sensitivity, requires fewer labeled antibodies, and is more economical. Indirect ELISA also provides greater flexibility, because different primary antibodies can be used with a single-labeled secondary antibody.


 

-Disadvantages of Indirect ELISA


Disadvantages of indirect ELISA: There is the possibility of cross-reaction (enzyme-labeled secondary antibody directly binds to the antigen), which may increase the background. At the same time, compared with direct ELISA, indirect ELISA experiment has more secondary antibody incubation steps, and the experiment cycle is prolonged.

The key to the success of the indirect method is the purity of the antigen. Although sometimes coating with crude antigen can achieve practical results, it should be purified as much as possible to improve the specificity of the test. Particular attention should be paid to removing impurities that can react with the serum of ordinary healthy people. For example, recombinant antigens with E. coli as engineered enzymes, if they contain E. coli ingredients, are likely to interact with anti-E. coli in the serum of people who have been infected with E. coli. Coli antibody reacts. Antigens should not contain substances that react with enzyme-labeled anti-human Ig, such as antigens from human plasma or human tissues. If the Ig is not removed, false positive reactions will occur in the test. In addition, if the antigen contains irrelevant proteins, it will also affect the coating effect due to competitive adsorption.

Another interference factor in the indirect method is the high concentration of non-specific antibodies contained in normal serum. The specific IgG tested in the patient’s serum accounts for only a small part of the total IgG. IgG has strong adsorptivity, and non-specific IgG can be directly adsorbed to the solid-phase carrier, and sometimes it can also be adsorbed to the surface of the coated antigen. Therefore, in the indirect method, the antigen is usually coated with an unrelated protein (such as bovine serum albumin) again to block the empty space on the solid phase. In addition, the specimen must be diluted first (1:40~1:200) during the testing process to avoid excessive negative background from affecting the judgment of the results.

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