Purification of Antibodies Using Affinity Chromatography

Abstract

Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilised to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently proteins G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics. This chapter is designed to inform the researcher about the basic techniques involved in the affinity chromatography-based purification of monoclonal, polyclonal, and recombinant antibodies. Examples are provided for the use of proteins A and G. In addition, tables are provided that allow the reader to select the most appropriate protein for use in the isolation of their antibody.

Perspectives on Immunoglobulins in Colostrum and Milk

“…These include traditional methods of ammonium sulfate precipitation and column chromatography [3,5,145,184]. Affinity chromatographic methods used to isolate IgG include lectins [185]; protein A or G chromatography [186,187], and more recently, isolation with protein A/G immobilized electrospun polyethersulfone membranes [188]; metal chelate chromatography [189,190]; and adsorption with polyanhydride microparticles [191]. The range of detection and quantification methods for IgG, most often analyzed by radial-immunodiffusion [192] or enzyme-linked immunosorbant methods [193], are now expanding to include methods that detect multiple proteins, such as thermally addressed immunosorbant assays [194], and rapid methods that may be integrated into milking systems, such as surface plasmon resonance-based immunosensors [195].…”

Development of a human whole blood assay for prediction of cytokine release similar to anti-CD28 superagonists using multiplex cytokine and hierarchical cluster analysis

“…The drawback to using a bacterial protein to immobilize mAbs is that the bacterial antigen itself may cause release of cytokines from monocytes and lymphocytes in vitro [33,46]. In addition there are slight differences in the amount of human or mouse IgG Protein A binds [47,48]. However, since we are using a concentration of beads that should produce maximal cytokine production, we believe that this is unlikely to be an issue.…”

The Purification of Natural and Recombinant Peptide Antibodies by Affinity Chromatographic Strategies

“…IgG isolation has been widely performed by using protein A, G, and L, which are the most popular immunoglobulin-binding proteins through their binding to the fragment crystallizable (Fc) or Fab region [ 3 ].…”