Immunoaffinity Chromatography: A Review

Affinity chromatography is a high resolution, high capacity, and one of the most powerful and diverse methods for separating proteins and other biological molecules of interest on the basis of a highly specific, reversible biological interaction between two molecules: an affinity ligand attached to a solid matrix to create a stationary phase, and a target molecule in a mobile phase. Specifically, immunoaffinity chromatography (IAC) relies on a solid stationary phase consisting of an antibody coupled to a chromatographic matrix or to magnetic beads, and harnesses the selective and strong binding of antibodies to their targets (Hage, 1998). Accordingly, any molecule that can be bound effectively by an antibody can be purified using IAC (Lesney, 2003). Purified antibodies are coupled to the inert solid phase and mixed with the antigen solution under conditions that favor adsorption. Following antigen capture, unwanted antigens are removed by washing, and the purified antigen is released by switching to conditions that favor desorption. Purification (often greater than 1000-fold) and simultaneous concentration of the target protein are thus achieved (Fitzgerald et al., 2011). One of the first uses of IAC was reported in 1951 by Campbell et al. who used immobilized bovine serum albumin on p-aminobenzyl cellulose to purify antialbumin antibodies. Since then, there has been a great expansion in the applications of IAC for analytical, clinical, and diagnostic purposes.