title: Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library.
abstract: 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.
authors.affiliation: Pacific Northwest National Laboratories, Biological Sciences Division, 902 Battelle Blvd., P.O. Box 999, Richland, WA 99352, USA. Michael.Feldhaus@pnl.gov
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Feldhaus
authors.forename: Robert W
authors.initials: RW
authors.lastname: Siegel
authors.forename: Lee K
authors.initials: LK
authors.lastname: Opresko
authors.forename: James R
authors.initials: JR
authors.lastname: Coleman
authors.forename: Jane M Weaver
authors.initials: JM
authors.lastname: Feldhaus
authors.forename: Yik A
authors.initials: YA
authors.lastname: Yeung
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
authors.forename: Peter
authors.initials: P
authors.lastname: Heinzelman
authors.forename: David
authors.initials: D
authors.lastname: Colby
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Swers
authors.forename: Christilyn
authors.initials: C
authors.lastname: Graff
authors.forename: H Steven
authors.initials: HS
authors.lastname: Wiley
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular; D005240:Feasibility Studies; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008832:Microchemistry / Q000379:methods; D008863:Microspheres; D036103:Nanotechnology / Q000379:methods; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins