Fluorescent antibody techniques

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An immunoblot

An immunoblot done with a crude Brucella ovis antigen and sera from infected and non-infected sheep. Those sera with clearly stained bands of 29 kDa (sera 5, 7, 11,and 13) are positive for Brucella ovis antibody

Term: 2014
Published: February 27, 2014
Revised: April 8, 2014

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
     

About The Instructor

Prof Moritz van Vuuren

Prof Moritz van Vuuren

  • BVSc MMedVet (Micro)(Pret)
  • Professor in Virology in the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa.
Creative Commons Attribution 4.0 International
This Work, Fluorescent antibody techniques, by Dr RW Worthington, Prof Moritz van Vuuren is licensed under a Creative Commons Attribution license.