UNIT 8.2 Immunoaffinity Chromatography

Abstract

This unit describes the isolation of soluble or membrane-bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high-pH or low-pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl β-D-glucoside can be used instead of Triton X-100 for elution. Because octyl β-D-glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.