Monoclonal Antibody Affinity Chromatography

Abstract

Affinity chromatography is one of the most convenient methods of purifying biological molecules, with many wide ranging applications (1, 2). The main prerequisite is to have a ligand that specifically binds to the target molecule. This ligand must also have two key properties other than exquisite specificity: It must be capable of being immobilized in an active form to a solid matrix, and the ligand/target complex must be readily dissociable under conditions that release the target molecule in an active form. These criteria are often best fulfilled for protein targets by antibody ligands. Monoclonal antibodies are the ligands of choice for most purposes, especially when a protein of unknown properties is being characterized. Monoclonal antibodies fulfill all the above criteria and also afford the advantage of being available in essentially unlimited supply. Moreover, they are themselves readily purified, often by affinity chromatography. The main disadvantage of monoclonal antibody ligands is that they must possess affinities that are sufficiently high to allow efficient purification and sufficiently low to allow efficient elution. This balance cannot really be tailored, but must be obtained by screening a number of specific monoclonals until empirically one is obtained with the desired properties. Once an appropriate monoclonal antibody has been identified, then in principle the purification of the corresponding antigen is straightforward and is limited in quantity only by the availability of the starting material.