Purification of antibodies using affinity chromatography.
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.
School of Biotechnology, Dublin City University, Dublin, Ireland.
Elaine
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Darcy
Paul
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Leonard
Jenny
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Fitzgerald
Martin
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Danaher
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O'Kennedy
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D051379:Mice; D009419:Nerve Tissue Proteins / Q000276:immunology; D013205:Staphylococcal Protein A / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C029152:G-substrate; C055047:Ig L-binding protein, Peptostreptococcus; D008024:Ligands; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A; D000645:Ammonium Sulfate