Fluorescent antibody techniques

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Fluorescent antibody (FA) tests, immune-enzyme assays and radio-immunoassays are primary binding assays. This implies that antigen-antibody complexes are formed during the test procedures, followed by detection or measurement of the amount of immune complexes formed.

Antibodies linked to fluorescent markers or enzymes can be used to detect antigen in tissues or tissue cultures or for the detection of antibody in sera by means of indirect tests. Fluorescein isothiocyanate is readily coupled to proteins and is the marker most commonly used for marking antibodies. When exposed to ultraviolet light, fluorescein radiates longer wavelength apple green coloured light. Sections or smears of tissues may be stained with fluorescein labelled antibodies and examined under an ultraviolet light microscope to locate antigenic material to which the antibody has attached.

When enzyme-linked antibodies are used to locate antigen, the section or smear is flooded with antibody coupled to an enzyme. Alternatively an uncoupled positive serum can be used and a second antibody coupled to an enzyme can be used to identify the attached antibody in a sandwich test. Sections are then washed and flooded with a substrate, which undergoes a chemical reaction catalysed by the enzyme. The substrate changes colour and is deposited in the tissues. Stained areas can then be visualised by conventional light microscopy.

The following topics will be covered in this sub-module:

  • Immunofluorescence assays
  • Immunoblotting and immunohistochemistry
License Condition: Creative Commons: Attribution 4.0  
Education Level: 
Continuing Professional Development (CPD)
Academic Year: 

Prof Moritz van Vuuren

  • BVSc MMedVet (Micro)(Pret)
  • Professor in Virology in the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa.