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, 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 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.