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Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

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  • Peter Durand Skottrup
  • Paul Leonard
  • Jakub Zbigniew Kaczmarek
  • Florian Veillard
  • Jan J. Enghild
  • Richard O'Kennedy
  • Aneta Sroka
  • Rasmus Prætorius Clausen
  • Jan Potempa
  • Erik Riise
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Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.

Original languageEnglish
JournalAnalytical Biochemistry
Volume415
Issue number2
Pages (from-to)158-67
Number of pages10
ISSN0003-2697
DOIs
Publication statusPublished - 15 Aug 2011

    Research areas

  • Adhesins, Bacterial, Amino Acid Sequence, Antibodies, Bacterial, Bacteroidaceae Infections, Biological Markers, Cysteine Endopeptidases, Humans, Molecular Sequence Data, Peptide Library, Porphyromonas gingivalis, Protein Binding, Protein Isoforms, Recombinant Proteins, Saliva, Single-Chain Antibodies

ID: 44863385