Immunofluorescence Technique

Immunofluorescence technique (immunofluorescence technique) is also known as fluorescent antibody technique. Founded in the early 1940s, in 1942, Coons et al. reported several times that antibodies were labeled with fluorescein isocyanate. It is a technology established on the basis of immunology, biochemistry and microscopy. Immunofluorescence technology includes fluorescent antibody technology and fluorescent antigen technology, because fluorescent pigments can not only bind to antibody globulin to detect or locate various antigens, but also bind to other proteins to detect or locate antibodies, but in actual work Fluorescent antigen technology is rarely used, so people are used to calling it fluorescent antibody technology, or immunofluorescence technology.
The main characteristics of this technology are: strong specificity, high sensitivity and fast speed. The main disadvantages are: the problem of non-specific staining has not been completely solved, the objectiveness of the result judgment is insufficient, and the technical procedures are still relatively complicated.

The Basic Principle of the Experiment

The basic reaction of immunology is the antigen-antibody reaction. Because the antigen antibody response is highly specific members of the opposite sex, so when an antigen-antibody reaction occurs, as long as one of the factors is known, the other factor can be detected. The immunofluorescence technique is to label the antibody (or antigen) with a fluorescent pigment that does not affect the activity of the antigen and antibody, and after combining with the corresponding antigen (or antibody), a specific fluorescence reaction is displayed under a fluorescence microscope.

Experimental Method

Direct Fluorescent Antibody Technique

This is the simplest and basic method of fluorescent antibody technology. Add fluorescent antibody dropwise to the specimen to be tested, and observe under a fluorescence microscope after reaction and washing. If the corresponding antigen exists in the specimen, it specifically binds to the fluorescent antibody, and the fluorescent antigen-antibody complex can be seen under the microscope. The advantages of this method are simplicity and specificity. But its disadvantage is that it needs to prepare corresponding specific fluorescent antibody to check each antigen, and the sensitivity is lower than that of the indirect method.

Indirect Fluorescent Antibody Technique

According to the principle of anti-globulin test, the method of labeling anti-globulin antibodies with fluorescein (labeled anti-antibody for short). The detection process is divided into two steps: the first time, the antibody to be tested (the first antibody) is added to the specimen sheet containing the known antigen for a certain period of time to wash away the unbound antibody. Second, drop the labeled anti-antibody. If the antigen-antibody in the first step has been combined, the labeled anti-antibody added at this time will bind to the antibody (primary antibody) molecule that has been immobilized on the antigen to form an antigen-antibody-labeled anti-antibody complex and display specific fluorescence . The advantage of this method is that the sensitivity is higher than that of the direct method, and it can be used to detect multiple antigen-antibody systems of the same animal without preparing a fluorescein-labeled anti-globulin antibody. Staphylococcus a protein can also be labeled with fluorescein. Instead of labeled anti-globulin antibody, it is used for indirect fluorescent staining, and is not limited by the species of the primary antibody source, but the sensitivity is lower than the labeled anti-antibody method. The indirect method is sometimes prone to produce non-specific fluorescence, which is its disadvantage. This method is often used for the detection of various autoantibodies. If the primary antibody is known, the indirect method can also be used to identify the unknown antigen.
Because immunoglobulin is species-specific, the labeled anti-globulin antibody must be prepared by immunizing other animals with animal serum globulin of the same species as the primary antibody.
The advantage of the indirect staining method is that it can detect unknown antigens and unknown antibodies; a labeled anti-globulin antibody can be combined with the antibodies of all animals of the same species to detect various unknown antigens or antibodies. High sensitivity. The disadvantages are: because there are many factors participating in the reaction, the possibility of interference is also greater, the judgment result is sometimes difficult, the operation is cumbersome, the comparison is more, and the time is long. The indirect method should be set to control negative and positive specimens, as well as an intermediate layer control (ie, intermediate layer plus negative serum instead of positive serum).

Anti-complement Staining Method

Anti-complement dyeing fish method is referred to as complement method, which is an improved method of indirect fish dyeing method. Founded by Goldwasser et al. This method uses the principle of complement fixation reaction to label anti-complement antibodies with fluorescein to identify unknown antigens or unknown antibodies (serum to be tested). The staining procedure is also divided into two steps: first add unlabeled antibodies and complement to the antigen specimen, make it react, wash with water, and then add labeled anti-complement antibodies. If the antigen-antibody reacts in the first step to form a complex, the complement is bound by the antigen-antibody complex, and the fluorescein-labeled anti-complement antibody added in the second step specifically reacts with the complement to form an antigen-antibody -Complement-anti-complement antibody complex, which emits fluorescence.
The anti-complement staining method has the same advantages as the indirect method, in addition, it also has its unique advantages: that is, only one labeled anti-complement antibody is needed to detect various antigen-antibody systems. Because the role of complement is not specific, it can react with the antigen-antibody system of thousands of mammals. Its disadvantage is that there are many ingredients involved in the reaction, and the dyeing procedure is more complicated and troublesome.
In addition to the above three methods, there are also some methods developed on this basis, such as two-layer method, sandwich method, mixed method, three-layer method, antibody-anti-complement method and so on.

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Indirect Fluorescent Antibody Test