High efficiency recovery and epitope-specific sorting of an scFv yeast display library

In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library (Feldhaus et al., 2003), one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs(TM) system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor. (C) 2004 Elsevier B.V. All rights reserved.