Immunofluorescence (IF) is a technique of detecting specific target proteins, antigens etc. by using fluorescent-labeled antibodies. It is based on use of specific antibodies which have been chemically conjugated to fluorescent dyes which binds directly or indirectly to cellular antigens.
These tests are of two types:
- Direct/Primary immunofluorescence test.
- Indirect/ Secondary immunofluorescence test.
Indirect immunofluorescence test
Secondary immunofluorescence is a double-layer method in which two antibodies are used.
- Primary antibody (Unlabeled) which specifically binds target molecule.
- Secondary antibody (labeled with fluorophore) whichrecognizes primary antibody and binds to it.
Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing number of fluorophore molecules per antigen.
Steps in indirect Immunofluorescence
Indirect immunofluorescence is a two-stage process.
- First stage: Incubation of a fixed antigen (e.g., in a cell or tissue) with unlabeled antibody, which becomes associated with antigen. Patient’s serum to be tested is applied to the slide, followed by careful washing. If serum contains antibody against an antigen, it will combine with it.
- Second stage: Addition of fluorescent dye-labeled antibody which binds antibody-antigen complex. Fluorescence occurs which is observed by a fluorescence microscope.
Uses of indirect Immunofluorescence
- Detection of specific antibodies for sero-diagnosis of syphilis, leptospirosis, amoebiasis, toxoplasmosis, and many other infectious diseases.
- Enumeration and identification of lymphocyte sub populations by employing monoclonal antibodies and cytofluorographs.
- Identification of antibody classes by using fluorescent antibodies specific for different immunoglobulin isotypes.
- Detection of auto-antibodies, such as antinuclear antibodies in autoimmune diseases.
Advantages of indirect Immunofluorescence
- Use of single labeled anti-globulin (antibody to IgG) as a “universal reagent” to detect many different specific antigen–antibody reactions.
- More sensitive than direct immunofluorescence test.
- More flexible as variety of different secondary antibodies and detection techniques can be used for a given primary antibody.
Disadvantages of indirect Immunofluorescence
- Expensive fluorescence microscope and reagents
- Trained personnel are required to perform.
- Have a factor of subjectivity that may result in erroneous results.