- In this method, a great number of fluorescent dyes (e.g., fluorescein, rhodamine) are used as specific reporter labels.
- As a typical example, fluorescein isothiocyanate (FITC) can be attached to known specific antibodies.
- This labelled antibody is used to identify epitopes on the surfaces of complex corpuscular antigens (microbial and eukaryotic cells, erythrocytes, etc.).
- Dye, covalently attached to antibody molecules, becomes visible by irradiation in the luminescent microscope.
- In another case, dye-labelled antiglobulin antibody may be employed to reveal the presence of a specific antibody in patient or animal serum.
- These tests are quick, accurate and readily performed. They are divided into two distinct types: direct and indirect immunofluorescence tests.
Direct immunofluorescence tests.
- In the first case of a direct reaction, the microorganism to be tested is fixed to a slide. The known antibodies labelled with fluorescein isothiocyanate (FITC) are added to the slide.
- They should interact directly with unknown microbial antigen. After short-term incubation, the slide is washed thoroughly.
- Antibodies fixed upon the microbial antigens remain to hold on the slide. Then the specimen is examined by luminescent microscopy with excitation by violet light.
- If the microorganisms correspond to the antibody, they will be surrounded with bright yellow-green halo of fluorescence.
Indirect fluorescent antibody test
- It is used in both directions: for identification of unknown bacterial cells or for estimation of specific antibodies in patient’s serum.
- In the latter case, the known microorganism is fixed on the slide, and a sample of the unknown serum is added to it.
- After incubation, the slide is washed. If the unknown serum antibodies match the antigen, they remain fixed to it.
- The attached unknown antibody is to be detected by the next treatment with a fluorescent-labelled antiglobulin antibody (anti-human gamma globulin).
- Finally, the slide is examined by luminescent microscopy.
- When viewed with violet light, the complex appears yellow-green.
- The indirect test is often more sensitive than the direct one because the larger amounts of labelled antibodies absorb by antigen molecules.
- Moreover, the labelled antiglobulin is a “universal reagent” that binds to all antibodies that pertain to the same species.
Both types of immunofluorescence reactions are employed widely in immunology and microbiology. They are used in microbial identification, cell typing, for detection of virus-infected cells, etc.
The indirect assay is available for serological diagnosis of Lyme disease, Q fever, syphilis and many other pathological processes, where specific antibodies to infectious agents arise.