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Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

Journal Article · · PLoS ONE
 [1];  [2];  [2];  [2];  [2];  [2]
  1. Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); DOE/OSTI
  2. Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
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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. 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. 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. 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.
Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Laboratory Directed Research and Development (LDRD) Program; Department of Homeland Security
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1627484
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 12 Vol. 6; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

References (16)

Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. journal February 1994
Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy journal December 2004
Protein production by auto-induction in high-density shaking cultures journal May 2005
Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries journal July 2001
Use of a heterodimeric coiled-coil system for biosensor application and affinity purification journal September 1998
Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils:  Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding † journal February 2003
Genome sequence of Yersinia pestis, the causative agent of plague journal October 2001
Exploiting recombination in single bacteria to make large phage antibody libraries journal January 2000
Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis journal November 1999
Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context journal September 2006
Avirulent Strains of Pasteurella Pestis journal September 1943
Eliminating helper phage from phage display journal November 2006
Yersinia pestis--etiologic agent of plague. journal January 1997
Factors promoting acute and chronic diseases caused by yersiniae. journal July 1991
Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen journal December 1998
Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype journal April 2008

Cited By (8)

Multivalent pIX phage display selects for distinct and improved antibody properties journal December 2016
Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B journal July 2014
Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands journal September 2016
Parallelized Microscale Expression of Soluble scFv book January 2019
Principles and application of antibody libraries for infectious diseases journal September 2014
Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding journal March 2018
Phage display—A powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications journal December 2012
Phage display—A powerful technique for immunotherapy: 2. Vaccine delivery journal December 2012

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
bacteriophages
cloning
enzyme-linked immunoassays
flow cytometry
lysozyme
phage display
yersinia pestis
yersinia pseudotuberculosis