The principle of immunofluorescence cytochemical method is to label the known antigen or antibody with fluorescein according to the law of antigen-antibody reaction to make fluorescent antigen or antibody, and then use it as a probe to detect the corresponding substances in tissues or cells. The specific types of methods include direct method, indirect method, sandwich method and complement method. Under a fluorescence microscope, the source, nature and location of the detected substance can be determined according to the fluorescence emitted by the complex formed.
Indirect Fluorescent Antibody is to use a specific antibody to react with the corresponding antigen in the specimen, and then use a fluorescein-labeled secondary antibody (anti-antibody) to bind to the primary antibody in the antigen-antibody complex, and observe the specificity under a fluorescence microscope after washing. Fluorescence to detect unknown antigens or antibodies. Compared with the direct method, the sensitivity of this method is increased by 5-10 times, and one fluorescent secondary antibody can detect a variety of antigen-antibody systems, but the disadvantage is that it is easy to generate non-specific fluorescence. 
If you have a primary antibody (fluorescein-labeled) against the antigen to be tested, and your antigen to be tested is also abundantly expressed, it is not a bad idea to do the direct method. However, if you do not have the above two conditions, it is recommended that you do the Indirect Fluorescent Antibody (IFA) Test. For example, cytoskeletal proteins can be detected by direct immunofluorescence method; if the protein expression abundance is relatively low, direct method detection is more difficult, and fluorescence-labeled secondary antibodies need to be used for signal amplification to detect.
IFA is an assay which uses fluorescent microscopy to detect antibodies to specific antigenic material. This test is often used to confirm positive results obtained by ELISA(Enzyme Linked Immunosorbent Assay) or MFIA(Multiplexed Flurometric ImmunoAssay).It is typically used as a confirmation test as the location of antibody-antigen reactions can be visualized within an infectef cell. Advantages and Disadvantage of Indirect Fluorescent Antibody (IFA) Test
Advantages
❖Inexpensive to perform
❖The morphology and location of fluorescence can be evaluated to differentiate from non-specific reactions
Disadvantages
❖Fluorescent microscope is required.
❖Due to efficiency limitations, IFA is not primary serological screening tool.
❖Interpretation is subjective.
❖Results are not quantiative.
❖Limited to one antigen per slide.
❖Nonspecific fluorescence is common and intensity of fluorescence is common and intensity of fluorescence is variable.
❖As with all serological tests, IFA does not detect the infectious organism. IFA only provides a historical indication of infection (antibodies).
❖As they do not produce antibodies, IFA is not suitable for use with immunodeficient animals.
What is the Difference Between Direct and Indirect Fluorescent Antibody (IFA) Test
Using conjugated secondary antibodies to detect primary antibodies adds additional steps. Secondary antibodies are relatively inexpensive compared to primary antibodies. Using the same conjugated secondary antibody to detect different primary antibodies provides further cost savings. In indirect methods, appropriate secondary antibodies must be selected, adding complexity. This is especially true in multicolor experiments that require the use of multiple secondary antibodies, each targeting a different species and conjugated to a different dye. The availability of different conjugated secondary antibodies greatly increases flexibility. Multiple secondary antibodies combined with the primary antibody can amplify the signal. Secondary antibodies may cross-react with organisms that are not the target. Use of a pre-adsorbed secondary antibody prevents cross-reactivity. In the indirect method, samples containing endogenous immunoglobulins may show high background.
The Basic Principle of Indirect Antibody Test
The staining procedure is divided into two steps. In the first step, an unknown unlabeled antibody (specimen to be tested) is added to a known antigen sample, incubated at 37°C for 30 min in a wet box to fully bind the antigen and antibody, and then washed to remove the unlabeled antigen. bound antibody.
In the second step, fluorescently labeled antiglobulin antibodies or anti-IgG and IgM antibodies are added. If an antigen-antibody reaction occurs in the first step, the labeled antiglobulin antibody will further bind to the antigen-bound antibody, so that the unknown antibody can be identified.
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