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Advances in the Development of Phage-Based Probes for Detection of Bio-Species.

ABSTRACT: Bacteriophages, abbreviated as "phages", have been developed as emerging nanoprobes for the detection of a wide variety of biological species, such as biomarker molecules and pathogens. Nanosized phages can display a certain length of exogenous peptides of arbitrary sequence or single-chain variable fragments (scFv) of antibodies that specifically bind to the targets of interest, such as animal cells, bacteria, viruses, and protein molecules. Metal nanoparticles generally have unique plasmon resonance effects. Metal nanoparticles such as gold, silver, and magnetism are widely used in the field of visual detection. A phage can be assembled with metal nanoparticles to form an organic-inorganic hybrid probe due to its nanometer-scale size and excellent modifiability. Due to the unique plasmon resonance effect of this composite probe, this technology can be used to visually detect objects of interest under a dark-field microscope. In summary, this review summarizes the recent advances in the development of phage-based probes for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays, and highlighting the commonly used editing technologies of phage genomes such as homologous recombination and clustered regularly interspaced palindromic repeats/CRISPR-associated proteins system (CRISPR-Cas). Finally, we discuss the possible scenarios for clinical application of phage-probe-based detection methods.

SUBMITTER: Paramasivam K

PROVIDER: S-EPMC8773867 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Advances in the Development of Phage-Based Probes for Detection of Bio-Species.

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