What is an antibody?
Antibodies (also called immunoglobulins, or Igs) are specialized proteins that allow the immune system to distinguish between “self” and “non-self” proteins or polysaccharides. Antibody molecules comprise four linked polypeptide chains: two “heavy chains” and two “light chains” that are connected by disulfide bonds (Fig. 1). The amino acid sequence of the antigen-binding site is variable, allowing each antibody to recognize a unique epitope (a particular location within an antigen). Because of their specificity, antibodies can be used to detect the presence of specific biomolecules (i.e. peptides, proteins, antigens and hormones) in a complex sample.
How are the antibodies used for research made?
Antibodies are produced when animals (i.e. rabbits, mice and guinea pigs) are injected with an antigen. Since many different immune cells within the animal produce antibodies in response to the antigen, the serum will contain a mixture of antibodies that vary in their ability to bind the antigen. This mixture of antibodies is called polyclonal. If we isolate and culture individual immune cells from these animals, we can create a monoclonal antibody that recognizes a single epitope.
How are antibodies used in the laboratory?
Some uses of antibodies include:
- The Western blot (or immunoblot) uses antibodies to detect the presence of a protein in a mixed sample.
- Ouchterlony double diffusion is used to determine whether an antibody will react with a particular antigen.
- Radial immunodiffusion is used to determine the relative concentration of an antigen.
- The Enzyme Linked ImmunoSorbent Assay (ELISA) is an extremely sensitive technique that detects the quantity of antigens within a sample.
What are some concerns when using antibodies?
To be used in the laboratory, antibodies must have a specific, robust and reproducible interaction with their antigen. Antibodies that have a high affinity for non-specific antigens will have unwanted cross-reactions that can result in high backgrounds. In contrast, an antibody with a weak affinity may not be sensitive enough for antigen detection. These antibodies would produce results with a high false-positive or false-negative rate.