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Direct and Indirect Fluorescent Antibody Technology

2021-10-27
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Direct Fluorescent Antibody Technology

The direct method is the simplest and most basic method of fluorescent antibody technology. It uses the labeled antibody to directly bind to the corresponding antigen (antigen specimen to be tested) to identify the unknown antigen.

Advantages:

① Since there are only two factors involved in the reaction, the result judgment is relatively simple;

②Strong specificity, less cross-staining with other antigens;

③The operation steps are few, the method is simple and time-saving.

Disadvantages:

①Poor sensitivity;

②One labeled antibody can only identify one antigen;

③It cannot be used to identify unknown antibodies.


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Indirect Fluorescent Antibody Technology

The indirect method is currently the most commonly used method. First, use a known unlabeled antibody (primary antibody) to react with the antigen to be tested or an unknown antibody to react with a known antigen. After reacting for a certain period of time, wash off the unbound antibody, and then react with the labeled anti-immunoglobulin antibody ( Secondary antibody) response. In the first reaction, if the antigen-antibody reacts, the antibody is immobilized on the specimen, then the labeled antibody (secondary antibody) in the second reaction must react with the antibody in the antigen-antibody complex formed in the first reaction A reaction occurs so that the unknown antigen or antibody in the specimen can be identified by the trace of the secondary antibody.

Advantages:

①High sensitivity, 5-10 times that of direct method;

②With one kind of labeled antibody, multiple unknown antigens or antibodies can be identified in conjunction with more than one corresponding antibody or antigen;

③It can identify both unknown antigen and unknown antibody.

Disadvantages:

① There are many factors involved in the reaction, which are prone to non-specific staining, and sometimes it is difficult to judge the result;

② There are many operation steps and time-consuming.

Double Labeling

The dual labeling method uses two kinds of fluorescein (commonly used FITC and PE) to label antibodies with different specificities to detect different antigens in the same specimen. This method is often used in the study of cell surface antigens and receptors.

Related Articles:

Fluorescent Antibody Technique (Direct, Indirect)

Indirect Fluorescent Antibody Test

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