Immunoaffinity Chromatography (Molecular Biology)

Immunoaffinity chromatography (IAC) is a widely used method for the selective extraction of antibodies or other biomolecules on the basis of the biospecific affinity between an antibody (Ab) and its antigen (Ag) (1). The antigen may be a small molecule (a hapten) or a macromolecule (M) such as an enzyme, a hormone, a growth factor, and so on. IAC has been used for many years for the purification of antibodies by immobilized antigens and also for the purification of antigens (enzymes, receptors, hormones, growth factors, etc.) by antibodies anchored to inert carriers. The antibodies used can be polyclonal, provided that a pure antigen is injected to obtain the antibodies and that strict specificity is ascertained. It is often advisable to use anti-peptide (sequence-oriented) polyclonal antibodies to reduce the danger of heterogeneity in the specificity of the antibody population. Monoclonal antibodies are usually a very good choice because they are homogeneous and directed against a distinct epitope. Under ideal conditions, antibody columns allow separation of specific peptides or proteins from crude mixtures in one step. The procedure for an IAC purification involves the same steps described for affinity chromatography.

A major difficulty in the use of immobilized antibodies is the high affinity of some antibodies for their antigens. This sometimes makes the recovery of active enzymes difficult, because the harsh conditions that are required in the elution step bring about either an inactivation of the enzyme or the loss of an important regulatory function. Among the eluents that have (and are) being used in IAC are buffers with a low pH (2.2) or a high pH (11.5), chaotropes (5 M KSCN), or mild denaturants such as urea (3.5 to 8 M) or guanidinium salts (2-6 M). Therefore, attempts are sometimes made to purify by exclusion (also known as "reverse immunoadsorption"). In this procedure, the contaminating proteins are the ones to be adsorbed and removed, while the desired protein is excluded (2).

With the introduction of monoclonal antibodies in IAC, it became possible to use homogeneous immunoglobulins not only directed against a distinct antigenic determinant (3, 4) and to tailor their affinity. A lowered affinity permits milder conditions for elution and reduces the possibility of irreversible denaturation during the elution step. As expected, the use of monoclonal antibodies has had a significant impact in biochemistry, in molecular biology, and in medicine.

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