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Abstract
BackgroundMost 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.MethodsPhage 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.ResultsSeven 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.ConclusionsOur 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.
Highlights
Yersinia pestis is a gram-negative, non-spore-forming bacterium belonging to the family Enterobacteriaceae that is known to have evolved from the enteric pathogen Yersinia pseudotuberculosis approximately 20,000 years ago [1]
Phage display technology was used to select multiple single chain Fv antibody fragment (scFv) clones that bound to recombinant fraction 1 (F1) antigen
The initial aim of this study was to identify scFv antibodies that could be used for Y. pestis immunodetection using a recombinant form of one of the most common and species-specific surface antigens of this organism, F1 [8], as a selection target. scFvs were selected from a previously described large scFv phage display library [18] using recombinant F1 as the selection target. 252 clones from the third cycle of library panning were screened after recloning the scFvs as alkaline phosphatase (AP)-fused proteins
Summary
Yersinia pestis is a gram-negative, non-spore-forming bacterium belonging to the family Enterobacteriaceae that is known to have evolved from the enteric pathogen Yersinia pseudotuberculosis approximately 20,000 years ago [1]. Among the eleven true Yersinia species three are pathogenic to humans; Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolytica, while all others are harmless [2]. The infectious agent can be dispersed and transmitted via inhalation causing pneumonic plague, the least common but most virulent form, which has the potential to cause high rates of morbidity and mortality in humans. Y. pestis is listed as a National Institute of Allergy and Infectious Disease, Biodefense Category A Priority pathogen (http://www3.niaid.nih.gov/topics/BiodefenseRelated/Biodefense/ research/CatA.htm), and is viewed as a high-priority agent that poses a risk to national security because it is relatively easy to acquire from the environment, and can be effectively dried and converted into an aerosol form. F1 is encoded by the caf 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. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection
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