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When the body is attempting to fight off disease, it produces antibodies – large, Y shaped proteins whose primary purpose is to neutralize disease-causing antigens like viruses and bacteria. These proteins are produced primarily by blood plasma cells and are dispatched by the immune system when a threat arises to mark antigens for eventual destruction. Comprehensive study of antibodies can lead to the development of effective therapies, life-saving vaccines and more effective diagnoses to prevent the spread of disease.
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Affinity Purification of Antibodies
For effective antibody-antigen research to proceed, antibodies must be separated from plasma and other components in the blood. This process, known as antibody purification, allows researchers to use antibodies in a predefined quantity to ensure the accuracy of their research. Antibody purification allows for easily reproducible results and reduces the interference of unknown compounds.
The most common way to purify antibodies is through affinity purification. During affinity purification, a ligand is linked to a gel with covalent bonds. Researchers pass a sample fluid through the gel and isolate the antibodies, which link to the associated ligands. Then, a buffer solution is used to detach the antibodies and recover them in a purified form without the surrounding compounds.
Protein A and Protein G
Protein A is a cell wall protein produced by multiple strains of the Staphylococcus aureus bacteria and is a very effective ligand used to bind to human antibodies. In fact, it has four binding sites that can bind with high affinity to the Fc binding site on human antibody IgG and can be used for affinity purification of IgG. In addition, it binds with some variability to different forms of IgM, IgA IgD and IgG – including IgG1,2, and 4 – and can be used to differentiate between the different antibodies.
Protein G is a similar cell wall protein but is produced by strains of group G Streptococci. It binds to the same Fc region of IgG as Protein A. However, as it does not bind to IgM, IgA, or IgD, and it can be used to sort antibodies. Since most human antibodies show a greater affinity for Protein G than Protein A, it can often serve as a better purification protein.
Unlike Protein A and Protein G, Protein A/G is an artificially engineered protein. Protein A/G is designed to combine the properties of Proteins A and G and utilizes the binding profiles of both within a single protein. This fusion protein is also more stable than either Protein A or G and does not depend on pH.
Protein A/G can bind to all subclasses of human IgG, unlike either of its predecessors. It is also useful for binding and identification of unknown antibodies whose classes have not been predetermined by researchers. In particular, it is useful for isolating mouse IgG and provides a more reliable bond to both mouse and human antibodies for purification.
A/G Purification Possibilities
With the use of recombinant A/G protein for affinity purification of antibodies, researchers continue to produce quantities of highly purified antibody samples free from outside interference. Depending on the antibody, using A/G can eliminate a step in the purification process and allows for a more direct path to a defined quantity of the desired antibody. As a result, the purified antibodies can be stored more easily, studied more effectively, and be acquired more economically than with the use of Protein A or Protein G alone.