Antibody Characteristics
A key component in all immunoassays, including the ELISA formats, is an antibody (Fig 6).
Antibodies are special proteins made by immune system cells of mammals. They are made to bind specifically to foreign molecules that invade the mammal, allowing the animal to fight off disease. The specificity of an antibody is critical so that the animal attacks foreign molecules (antigens) but not its own. Because of this specificity, antibodies can be found, for example, that bind to the bacterial version of Roundup resistant EPSPS but not to the plant’s version of this same enzyme. Details of this herbicide resistance can be found at the online lesson, Inhibitors of Aromatic Amino Acid Biosythesis. Once a scientist has obtained antibodies for the protein of interest, they can develop their immunoassay test.
Figure 6 Illustration of an antibody.
There are three components of antibodies: heavy chain, light chain, and binding sites. Notice in Figure 7 that the binding sites reside at the top of the heavy and light chains. This is where the antigen (or in our case the target protein, such as the cryIA Bt protein) will bind to the specific antibody (Fig 8). As indicated in the Figure 8 diagram, the antibody’s binding strength is determined by the three-dimensional 'fit' and chemical interactions between it and the antigen. It is this strength, also referred to as 'affinity', which determines how sensitive the protein test is. A greater affinity results in a greater sensitivity, meaning the test can detect lower amounts of the protein in a sample and also more specifically bind to only that protein.
Figure 7 Location of the heavy chain, light chain and binding sites of an antibody.
Figure 8 Illustration of antibody-antigen interactions. Strength of binding is determined by 3-dimensional ‘fit’ and chemical interactions between antibody and antigen.