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.
Siegel
Robert W
RW
Pacific Northwest National Laboratory, MSIN: K4-12, 902 Battelle Boulevard, P.O. BOX 999, Richland, WA 99352, USA.
Coleman
James R
JR
Miller
Keith D
KD
Feldhaus
Michael J
MJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D004815:Epidermal Growth Factor / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods*; D019151:Peptide Library; D015003:Yeasts / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004815:Epidermal Growth Factor