Antigen Purification by Monoclonal Antibody Immunoaffinity Chromatography

Abstract

In the past, isolation of a pure protein demanded many hours to develop a purification protocol, which included one or more cycles of time-consuming chromatography, only to have a final product that was enriched rather than purified. The development of a purification procedure had to be repeated for each protein of interest. Almost as much time was spent on purification as characterization of the protein and reproducibility varied from batch to batch of starting material. After techniques to produce monoclonal antibodies (MAbs) were developed, purification of antigens has been facilitated with MAbs coupled to Sepharose 4B (Pharmacia Biotech Inc., Piscataway, NJ). MAb reagents coupled to this support matrix require reduced time to purify proteins. Taking advantage of the exquisite specificity afforded by each antibody results in a final purified product that binds specifically to that MAb. Using immunoaffinity chromatography, a single antigen can be separated from very complex mixtures (e.g., detergent-disrupted cell lysates made from tissue-culture cells), and whole blood and organs (e.g., thymus and spleen).