Direct and Indirect Fluorescent Antibody Technology

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 resulting 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 use to identify unknown antibodies.

Indirect Fluorescent Antibody Technology

The indirect method is currently the most commonly used. 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 responding for a certain period, wash off the unbound antibody and 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. The labeled antibody (secondary antibody) in the second reaction must respond with the antibody in the antigen-antibody complex formed in the first reaction. A reaction occurs so that the secondary antibody's trace can identify the specimen's unknown antigen or 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 novel antigens and unknown antibodies.

Disadvantages:

① There are many factors involved in the reaction, which are prone to non-specific staining, and sometimes it isn't easy to judge the result;

② There are many operation steps, and they are 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 foreign antigens in the same specimen. This method is often used in studying cell surface antigens and receptors.