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

Journal Article · Mon Mar 01 04:00:00 EST 2004 · Journal of Immunological Methods

DOI:https://doi.org/10.1016/j.jim.2004.01.005· OSTI ID:15007831

Siegel, Robert W; Coleman, James R; Miller, Keith D; Feldhaus, Michael

In order to more productively utilize the rich source of antigen specific reagents present in the previously described non-immune 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 MacsTm 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 3 epitope specific scFv (including a previously uncharacterized epitope) to a 6 kd protein, epidermal growth factor EGF.

• Research Organization: Pacific Northwest National Lab., Richland, WA (US)
• Sponsoring Organization: US Department of Energy (US)
• DOE Contract Number: AC06-76RL01830
• OSTI ID: 15007831
• Report Number(s): PNNL-SA-39887
• Journal Information: Journal of Immunological Methods, Journal Name: Journal of Immunological Methods Journal Issue: 1-2 Vol. 286
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIGENS
EFFICIENCY
GROWTH FACTORS
MONOCLONAL ANTIBODIES
SORTING
YEASTS