Journal of Immunological Methods 2009-01-01

A yeast display immunoprecipitation method for efficient isolation and characterization of antigens

Yong Ku Cho, Irene Chen, Xin Wei, Lingjun Li, Eric V. Shusta

Index: J. Immunol. Methods 341(1-2) , 117-26, (2009)

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Abstract

Yeast antibody display has found a wide variety of applications including antibody affinity maturation, epitope mapping, and library screening. Here we report a yeast display immunoprecipitation (YDIP) technique that employs yeast cells displaying single-chain antibody fragments (scFv) on their surface as affinity capture reagents to isolate and characterize antigens. We show that displayed single-chain antibody fragments are active in a variety of detergent solutions commonly used for immunoprecipitation and that the antigen–antibody interaction can be accurately quantified by YDIP coupled with flow cytometry. The YDIP method has also been optimized so that it is compatible with commonly used protein characterization tools such as Western blotting, silver staining, and mass spectrometry. From complex protein mixtures, we have used YDIP to isolate, analyze and sequence both soluble and plasma membrane antigens using tandem mass spectrometry. In the case of the membrane antigen, YDIP coupled with tandem mass spectrometry was successful in identifying neural cell adhesion molecule (NCAM) as the antigen for an antibody previously selected as binding to the plasma membranes of brain endothelial cells. The presented method therefore has potential to facilitate antibody-antigen characterization.