Fast and reliable diagnostic assays are required for a resilient detection of clinical infections or biothreat-relevant pathogens. While PCR has proven to be the gold standard for nucleic acid detection, the identification of pathogen particles is still challenging and depends on the availability of well-characterized, chemically stable, and selective recognition molecules. Here, we report the screening of a phage display random peptide library for vaccinia virus-binding peptides. The identified peptide was extensively characterized using peptide-probe ELISA, surface plasmon resonance, nLC-MS/MS, Western Blot, peptide-based immunofluorescence assay, and electron microscopy. Following identification, the phage-free, synthetic peptide, designated αVACVpep05, was shown to bind to vaccinia virus and other orthopoxviruses. We can demonstrate that the highly conserved orthopoxvirus surface protein D8 is the interaction partner of αVACVpep05, thus enabling the peptide to bind to other orthopoxviruses, including cowpox virus and monkeypox virus, viruses that cause clinically relevant zoonotic infections in humans. The process of phage display-mediated peptide identification has been optimized intensively, and we provide recommendations for the identification of peptides suitable for the detection of further pathogens. The peptide described here was critically characterized and seems to be a promising reagent for the development of diagnostic platforms for orthopoxviruses. We believe that our results will help to promote the development of alternative, nonantibody-based synthetic detection molecules for further pathogens.
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The authors are grateful to P. Wojciech Dabrowski for programming the “Library Insert Finder” software and to Andreas Kurth for providing us with adenovirus. We also thank Ursula Erikli for copy-editing. This work was funded within the BMBF/VDI-financed BiGRUDI network of the Robert Koch Institute (Berlin).
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