Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library

• DOI: 10.1038/nbt785
• Publication Date: 2003-01-30T21:35:23Z
• Authors: Michael J. Feldhaus; Robert W. Siegel; Lee K. Opresko; James R. Coleman; Jane M. Weaver Fe...; Yik A. Yeung; Jennifer R. Cochran; Peter Heinzelman; David Colby; Jeffrey Swers; Christilyn Graff; H. Steven Wiley; K. Dane Wittrup
• Keywords: Male; 0303 health sciences; Microchemistry; Immunoglobulin Variable Region; Membrane Proteins; Saccharomyces cerevisiae; Flow Cytometry; Polymerase Chain Reaction; Microspheres; Recombinant Proteins; 03 medical and health sciences; Peptide Library; Gene Expression Regulation, Fungal; Feasibility Studies; Humans; Nanotechnology; Female; Cloning, Molecular; Immunoglobulin Fragments; Cells, Cultured

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.