Immunofluorescence- Definition, Types, Steps, Uses

What is Immunofluorescence?

Immunofluorescence is an assay that is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies. The specificity of antibodies to their antigen is the base for immunofluorescence.

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. In the immunofluorescence test, a fluorescent dye that illuminates in UV light is used to detect/show the specific combination of an antigen and antibody. The dye usually used is fluorescein isothiocyanate, which gives yellow-green fluorescence. Immunofluorescence tests are also termed fluorescent antibody tests (FAT).


Fluorescent dyes, such as fluorescein isothiocyanate and lissamine rhodamine, can be tagged with antibody molecules. They emit blue-green and orange-red fluorescence, respectively under ultraviolet (UV) rays in the fluorescence microscope. This forms the basis of the immunological test. 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

A direct immunofluorescence test is used to detect the unknown antigens in a cell or tissue by employing a known labeled antibody that interacts directly with unknown antigens. If an antigen is present, it reacts with labeled antibody and the antibody-coated antigen is observed under UV light of the fluorescence. It involves the use of labeled antiviral antibodies.


The specimen is placed on the slide; fluorescent-labeled antibody is then added to it and allowed for some time for Antigen-Antibody reaction. The preparation is then washed which will allow the removal of other components except for the complex of antigen and fluorescent-labeled antibody. On microscopy (Fluorescence Microscopy), Antigen-Antibody complexes are observed fluorescing due to the dye attached to the antibody.

The need for the preparation of separately labeled antibodies for each pathogen is the major disadvantage of the direct immunofluorescence test.

Indirect immunofluorescence test

Indirect fluorescence is a double antibody technique. The unlabeled antibodies which have bound to the antigens are visualized by a fluorescent antiglobulin reagent directed at the unlabeled antibodies. The indirect immunofluorescence test is used for the detection of specific antibodies in the serum and other body fluids for the serodiagnosis of many infectious diseases.


Indirect immunofluorescence is a two-stage process.

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 antibodies against the antigen, it will combine with the antigen on the slide.

Second stage: The combination of antibody with antigen can be detected by the 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 an unlabeled antibody, which becomes associated with the antigen. After careful washing, a fluorescent antibody (e.g. fluorescent-labeled anti-IgG) is added to the smear. This second antibody will become associated with 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 often more sensitive than the direct immunofluorescence test.

Indirect immunofluorescence test is used widely to:

  1. Detect specific antibodies for serodiagnosis of syphilis, leptospirosis, amoebiasis, toxoplasmosis, and many other infectious diseases;
  2. Identify the class of a given antibody by using fluorescent antibodies specific for different immunoglobulin isotypes;
  3. Identify and enumerate lymphocyte subpopulations by employing monoclonal antibodies and cytofluorographs; and
  4. Detect autoantibodies, such as antinuclear antibodies in autoimmune diseases.

Immunofluorescence may also be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules. Immunofluorescence has been widely used in biological research and medical research.

The major limitation of immunofluorescence is that the technique requires

  1. expensive fluorescence microscope and reagents,
  2. trained personnel
  3. have a factor of subjectivity that may result in erroneous results

The biological samples include tissue and cells. Immunofluorescence aid to evaluate whether or not cells in a particular sample express the antigen in the study. In cases where an immune-positive signal is found, immunofluorescence also helps to determine which subcellular compartments are expressing the antigen. Immunofluorescence can be used on cultured cell lines, tissue sections, or individual cells.

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