• 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:

  1. Direct immunofluorescence test
  2. 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.

Direct fluorescent antibody test


  • 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.



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