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

Abstract

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/labelmore » 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.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1]
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  1. Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Laboratory Directed Research and Development (LDRD) Program; Department of Homeland Security
OSTI Identifier:
1627484
Grant/Contract Number:  
AC52-06NA25396
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 6; Journal Issue: 12; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; bacteriophages; enzyme-linked immunoassays; flow cytometry; yersinia pestis; phage display; cloning; lysozyme; yersinia pseudotuberculosis

Citation Formats

Lillo, Antonietta M., Ayriss, Joanne E., Shou, Yulin, Graves, Steven W., Bradbury, Andrew R. M., and Pavlik, Peter. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis. United States: N. p., 2011. Web. doi:10.1371/journal.pone.0027756.
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Lillo, Antonietta M., Ayriss, Joanne E., Shou, Yulin, Graves, Steven W., Bradbury, Andrew R. M., & Pavlik, Peter. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis. United States. https://doi.org/10.1371/journal.pone.0027756
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Lillo, Antonietta M., Ayriss, Joanne E., Shou, Yulin, Graves, Steven W., Bradbury, Andrew R. M., and Pavlik, Peter. Thu . "Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis". United States. https://doi.org/10.1371/journal.pone.0027756. https://www.osti.gov/servlets/purl/1627484.
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@article{osti_1627484,
title = {Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis},
author = {Lillo, Antonietta M. and Ayriss, Joanne E. and Shou, Yulin and Graves, Steven W. and Bradbury, Andrew R. M. and Pavlik, Peter},
abstractNote = {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.},
doi = {10.1371/journal.pone.0027756},
journal = {PLoS ONE},
number = 12,
volume = 6,
place = {United States},
year = {Thu Dec 08 00:00:00 EST 2011},
month = {Thu Dec 08 00:00:00 EST 2011}
}
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