- The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet light) and emitting them at a different wavelength (visible light) is known as fluorescence.
- Fluorescent dyes, such as fluorescein isothiocyanate and lissamine rhodamine, can be tagged with antibody molecules.
- They emit blue-green and orange-red fluorescence, under ultraviolet (UV) rays in the fluorescence microscope.
- Immunofluorescence tests have wide applications in research and diagnostics.
These tests are broadly of two types:
- Direct immunofluorescence test
- Indirect immunofluorescence test
Direct immunofluorescence test:
- Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by employing a known labeled antibody that interacts directly with unknown antigen.
- If antigen is present, it reacts with labeled antibody and the antibody coated antigen is observed under UV light of the fluorescence microscope.
- Direct immunofluorescence test is widely used for detection of bacteria, parasites, viruses, fungi, or other antigens in CSF, blood, stool, urine, tissues, and other specimens.
- The need for preparation of separate labeled antibody for each pathogen is the major disadvantage of the direct immunofluorescence test.
Indirect immunofluorescence test:
- The indirect immunofluorescence test is used for detection of specific antibodies in the serum and other body fluids for serological diagnosis of many infectious diseases.
- Indirect immunofluorescence is a two-stage process.
- In the first stage, a known antigen is fixed on a slide.
- Then the patient’s serum to be tested is applied to the slide, followed by careful washing.
- If the patient’s serum contains antibody against the antigen, it will combine with antigen on the slide.
- In the second stage, the combination of antibody with antigen can be detected by addition of a fluorescent dye-labeled antibody to human IgG, which is examined by a fluorescence microscope.
- The first step in the indirect immunofluorescence test is the incubation of a fixed antigen (e.g., in a cell or tissue) with unlabeled antibody, which becomes associated with the antigen.
- Next, after careful washing, a fluorescent antibody is added.
- The second antibody will become associated to the first, and the antigen–antibody complex can be visualized on the fluorescence microscope.
- The indirect method has the advantage of using a single labeled antiglobulin (antibody to IgG) as a “universal reagent” to detect many different specific antigen–antibody reactions.
- The test is more sensitive than the direct immunofluorescence test.
The major limitation of immunofluorescence is that the technique requires:
(a) Expensive fluorescence microscope and reagents.
(b) Trained personnel
(c) Have a factor of subjectivity that may result in erroneous results.