The operation of the basic method is as follows:
1. The antigen of the pathogen is coated in carbonate buffer at 4 ~ C overnight to form a solid phase antigen. After washing and removing the antigen that is not bound to the solid phase or not tightly bound, it is blocked with calf serum or bovine serum albumin, Wash to remove unbound parts and impurities.
2. Add a clinical sample containing the antibody to be tested, such as serum, and wash the plate after incubation for a certain period of time; at this time, the antibody to be tested will react with the specific antigen on Guyang and be adsorbed on the solid phase.
3. Add enzyme-labeled anti-human IgG antibody and wash the plate after incubation for a certain period of time; at this time, the solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex is formed on the solid phase.
4. Add enzyme substrate and incubate for color determination (Figure 2-6).
The most commonly used indirect methods for antibody detection are the determination of hepatitis C virus antibodies (anti-HCV), human immunodeficiency virus antibodies (anti-HIV), and Treponema pallidum antibodies. It can be seen from the above measurement mode that the antibody is measured indirectly. Strictly speaking, only the IgG class of the antibody is measured, and IgM and IgA classes are not involved, which is determined by the enzyme-labeled secondary antibody.
A major factor affecting the indirect method for measuring antibodies is the purity of the coated antigen. At present, the antigens used in the indirect method for detecting antibodies are generally genetically engineered recombinant antigens, such as HCV NS3, NS4, NS5, HIV gp41 and gpl20 and Treponema pallidum TpN15, TpN17, TpN47 and so on. When purifying genetically engineered antigens, try to remove the antigen of the host used for expression, such as E. coli, to avoid false positive reactions due to the presence of antibodies to this host antigen. In addition, due to the high concentration of IgG antibodies in the body, most of which are non-specific IgG produced by the body in contact with the external environment. Therefore, in order to avoid the false positive reaction caused by the adsorption of these high concentrations of non-specific IgG on the solid phase , Usually need to do a certain degree of dilution of the test sample.
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