Indirect method

The indirect method is a commonly used method for detecting antibodies. The principle is to use an enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect the test antibody bound to the solid phase antigen, so it is called the indirect method. The operation steps are as follows:

1) The specific antigen is connected with the solid phase carrier to form a solid phase antigen. Wash to remove unbound antigen and impurities.
2) Add diluted test serum and incubate for reaction. The specific antibodies in the serum bind to the solid-phase antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies are left on the solid phase carrier, and other components in the serum are washed away during the washing process.
3) Add enzyme-labeled anti-antibody. 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-labeled antibody antibody, thereby indirectly labeling the enzyme. After washing, the amount of enzyme on the solid phase carrier is positively correlated with the amount of antibody tested in the specimen.
4) Add substrate color

This method is mainly used for the diagnosis of infectious diseases by the detection of pathogen antibodies. The advantage of the indirect method is that as long as the coated antigen is changed, the same enzyme-labeled anti-antibody can be used to establish a method for detecting the corresponding antibody.
The key to the success of the indirect method is the purity of the antigen. Although coating with crude antigen can sometimes achieve practical and effective results, it should be purified as much as possible to improve the specificity of the test. Special attention should be paid to the removal of impurities that can react with the serum of general healthy people, such as recombinant antigens using E.Coli as an engineering enzyme. .Coli antibody reacts. Antigens must not contain substances that react with enzyme-labeled anti-human Ig, such as antigens from human plasma or human tissue. If Ig is not removed, false positive reactions will also occur in the test. In addition, if the antigen contains irrelevant protein, it will also affect the coating effect due to competitive adsorption.

Another interference factor in the indirect method is the high concentration of non-specificity contained in normal serum. The specific IgG detected in the patient's serum accounts for only a small part of the total IgG. IgG is very adsorbable, non-specific IgG can be directly adsorbed on the solid phase carrier, and sometimes can also be adsorbed on the surface of the coated antigen. Therefore, in the indirect method, the antigen is coated with an extraneous protein (such as bovine serum albumin) once more 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 excessively high negative background affecting the judgment of the results.

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