title: Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis.
abstract: BACKGROUND
  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 such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.
  
  
  METHODS
  Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.
  
  
  RESULTS
  Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.
  
  
  CONCLUSIONS
  Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Antonietta M
authors.initials: AM
authors.lastname: Lillo
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Ayriss
authors.forename: Yulin
authors.initials: Y
authors.lastname: Shou
authors.forename: Steven W
authors.initials: SW
authors.lastname: Graves
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
authors.forename: Peter
authors.initials: P
authors.lastname: Pavlik
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005453:Fluorescence; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015010:Yersinia pestis / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D007202:Indicators and Reagents; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies