PHAGE BASED ASSAYS FOR THE SCREENING OF SCFV BINDING TO YERSINIA PESTIS

Los Alamos Nat Secty-Los Alamos Nat Lab, Los Alamos NM

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species like Yersinia enterocolitica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody (scFv) phage display library using F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for the F1 antigen. We have identified a set of anti-F1 scFv that can be used as phage-displayed proteins for specific and convenient detection of Y. pestis. Binding to recombinant F1 was determined by one step ELISA and results compared to those obtained by multiplex flow cytometry, Although similar results were obtained from each method, we found that when analyzing affinity reagents, multiplex flow cytometry is a quicker, more convenient (high data quantity and low antigen consumption) and higher quality (wider dynamic range) alternative assay to ELISA.