Antibody Purification

Sometimes an immune response can produce antiserum or monoclonal hybridomas that may work well as unpurified antisera, ascites fluid, or culture supernatant. Commonly, affinity chromatography is used to clean up antibodies. Affinity chromatography directly uses the antibody’s ability to bind in a matrix format to enable the less tightly bound and nonspecific immunoglobulins to be washed away.

Commonly, antibodies are purified using protein A or protein G that is conjugated to a sepharose bead. Protein A and G are isolated from bacteria, and these proteins are used due to their intrinsic antibody-binding properties. These proteins bind the Fc regions of the antibodies. Different species of animal produce antibodies with better binding affinities to either protein A or G.

Antibodies may also be immunoaffinity purified. Immunoaffinity purification entails purifying the antibody against the immunogen, either the original peptide or protein, which is linked to a sepharose bead.