A yeast display immunoprecipitation method for efficient isolation and characterization of antigens.
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.
Cho
Yong Ku
YK
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Chen
Irene
I
Wei
Xin
X
Li
Lingjun
L
Shusta
Eric V
EV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001921:Brain / Q000276:immunology; D001923:Brain Chemistry / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology; D042783:Endothelial Cells / Q000737:chemistry* / Q000276:immunology; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry* / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000941:Antigens; D019006:Neural Cell Adhesion Molecules; D011994:Recombinant Proteins