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Immunofluorescence – Principle, Types, Applications, Advantages, Disadvantages

Immunofluorescence is an antigen-antibody reaction where the antibodies are tagged (labeled) with a fluorescent dye and the antigen-antibody complex is visualized using ultra-violet (fluorescent) microscope.

Fluorochrome is a dye that absorbs ultra-violet rays and emits visible light. This process is called fluorescence.

Commonly used fluorochrome are Acridine Orange, Rhodamine, Lissamine, and Calcofluor white. However, these fluorochromes are used for general fluorescence.

When fluorescein (FITC) is excited by a blue (wavelength 488nm) light, it will emit a green (520nm) color. Phycoerythrin (PE) emits an orange (570nm) color.

The fluorochrome commonly used in immunofluorescence are fluorescein isothiocyanate (green) and tetramethylrhodamine isothiocyanate.

Principle of Immunofluorescence

Fluorescent dyes (fluorochrome) illuminated by UV lights are used to show the specific combination of an antigen with its antibody.

The antigen-antibody complexes are seen fluorescing against a dark background. Immunofluorescence tests are referred to as fluorescent antibody tests (FAT).

Immunofluorescence - Principle, Applications, Advantages, Disadvantages

Types of fluorescent antibody tests:-

There are two types of fluorescent antibody tests (FAT): Direct and Indirect

1.) Direct fluorescent antibody tests

Direct FAT is used to detect and identify an unknown antigen in specimens, for example, Viral, bacterial, and parasitic antigens.

It is called direct test because the fluorescent dye is attached, or labeled, directly to the antibody.

The fluorochrome used is usually fluorescein isothiocyanate (FITC), which gives a yellow-green fluorescence.

A fluorescent substance is one that, when absorbing light of one wavelength, emits light of another (longer) wavelength.

The procedure of Immunofluorescence (An Example):

  1. A tissue or smear containing the organism (antigen) is fixed to a glass slide and incubated with the fluorescent antibody (antibody chemically linked to FITC).
  2. It is then washed to remove the unbound antibody.
  3. Examined by dark-field illumination under a microscope with a UV light source.
  4. The antigenic particles that have bound the labeled antibodies are seen to fluoresce brightly. Interpretation of the results:
  5. Presence of fluorescence: positive test for a particular antigen
  6. No fluorescence: negative or absence of particular antigen

Direct FAT can be used:

  • To identify bacteria when the numbers are very low,
  • To detect viruses growing in tissue culture or tissues from infected animals such as rabies virus in the brains of infected animals or antigens of HIV on the surface of infected cells.

Also Read: Enzyme-Linked Immunosorbent Assay (ELISA)

2.) Indirect fluorescent antibody test

In the indirect FAT, the unlabelled antibody combines with antigen and the antigen-antibody complex is detected by attaching a fluorescent-labeled anti species globulin to the antibody.

The antibody, therefore, is labeled indirectly. Fluorescent-labeled antihuman globulin is used if the antibody is of human origin.

The indirect FAT is used in two main ways

  • To detect and identify unknown antigen in specimens
  • To detect antibodies in a patient serum using a known antigen (microorganism).

Indirect FAT to detect the antigen

In this test, a slide preparation of the specimen is made and an unlabelled specific antibody is added.

After allowing time for the antigen and antibody to combine, the preparation is washed leaving the only antibody that has combined with the antigen on the slide.

A fluorescent-labeled anti- species globulin is added and allowed to combine with the antibody. The excess is washed from the slide.

The preparation is examined by fluorescence microscopy using the correct filters. The antigen-antibody complex will be seen fluorescing brightly.

Indirect FAT to detect Antibody

In this test, a known antigen is placed on the slide and the patient’s serum is added.

The preparation is then washed and fluorescent-labeled antihuman globulin is added to demonstrate the antigen-antibody reaction.

The preparation is examined by fluorescence microscopy using the correct filters.

Application of immunofluorescence test

They are widely used in the serological diagnosis of

  • bacterial,
  • viral,
  • fungal,
  • and parasitic diseases.

Advantages of immunofluorescence test

Clear positioning. This assay can give research on the clear subcellular localization of molecules. The expression of molecules can be observed directly.

High specificity. The preparation of the sample can effectively protect the natural structure of the antigen

High sensitivity. The target with low expression can be detected with immunofluorescence.

Easy to operate. The test result can get quickly. This assay can be used for in-vivo experiments.

Multiple staining. The multiple targets can be detected in the same sample.

Disadvantages of these tests are:

  • Special training is needed to perform and read Immunofluorescence tests.
  • Fluorescence microscopy and high-quality reagents are required.

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Science Direct


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