Imunofluorescence is a technique used to demonstrate structures and substances in cells and tissues. It relies on the tendency of antibodies to bind specifically to certain materials.Generalised structure of an Antibody

An antibody is a protein produced by cells in the immune system. It is made up of a number of subunits consisting of separate polypeptide chains. There are two major types of subunit – the heavy and light chains, and each single antibody has two light chains and two heavy chains.

Antibodies can be thought of as having a shape like a capital letter Y. Their functionality is determined by the variable regions, found at the ends of each of the branches of the Y. The variable regions are made up of sections of the heavy and light chains.

Antibodies work by binding to other substances called antigens through the variable region. The region of an antigen to which an antibody binds is called the epitope.

Each antibody produced has a variable region which specifically binds onto the epitope of a single antigen. This is what gives antibodies their specificity.

In the body, antibodies are produced as part of the humoral immune response. When cells in the body come into contact with an antigen, they “instruct” specialised B lymphocytes to make antibodies which can bind to that antigen – each line of cells produces just one type of antibody.

Once an antibody has been produced, it will bind to just that antigen. If the antigen forms part of a disease agent like a bacterium, parasite or virus, the antibody may assist in the body’s immune response by binding to that antigen and :

  • interfering with the agent’s ability to cause damage to the body (eg. antibodies binding to external proteins on a virus prevent those proteins from interacting with receptors on the outside of a host cell, preventing the cell from taking up the virus)
  • causing a phagocytic immune cell to engulf and destroy the disease agent
  • causing the disease agent to clump together and not be able to multiply or cause damage to the body
  • initiating destructive immune processes (eg. complement activation)

In addition to their role in immunity, antibodies are widely used in biological research and diagnosis because of their specificity. Antibodies can be mass produced in test animals by exposing those animals to antigens of interest and harvesting the cell lines which produce the antibody we are interested in. If a cell line produces just one antibody, the antibodies produced are called monoclonal antibodies. Polyclonal antibodies have a number of different binding affinities and come from multiple cell lines.

Immunofluorescence involves the use of antibodies which bind to cell components of interest, bound to a chemical substance which fluoresces under ultraviolet light (a flurophore). There are two main types of immunofluorescence :

  • Direct Immunofluorescence involves the use of a single labeled antibody. Because there is only one labeled antibody per antigen site, direct immunofluorescence gives a lower signal and is therefore less sensitive. Click here to see a diagram which shows the process of Direct Immunofluorescence.
  • Indirect Immunoflourescence involves the use of an unlabelled primary antibody which binds to the target antigen, and then a labeled secondary antibody which binds to the primary antibody. Because there may be multiple sites on the primary antibody for the secondary antibody to bind, the signal can be much stronger and indirect immunofluorescence is generally more sensitive than direct immunofluorescence. Click here to see a diagram which shows the process of Indirect Immunofluorescence.

This video from Professor Diane O'Dowd at the University of California - Irvine shows the process of indirect immunofluorescence (note : this link connects with an external site and is a You Tube video, which may not be available to some users).