title: Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.
abstract: 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.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark. pds@farma.ku.dk
authors.forename: Peter Durand
authors.initials: PD
authors.lastname: Skottrup
authors.forename: Paul
authors.initials: P
authors.lastname: Leonard
authors.forename: Jakub Zbigniew
authors.initials: JZ
authors.lastname: Kaczmarek
authors.forename: Florian
authors.initials: F
authors.lastname: Veillard
authors.forename: Jan Johannes
authors.initials: JJ
authors.lastname: Enghild
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
authors.forename: Aneta
authors.initials: A
authors.lastname: Sroka
authors.forename: Rasmus Prætorius
authors.initials: RP
authors.lastname: Clausen
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
authors.forename: Erik
authors.initials: E
authors.lastname: Riise
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D016866:Bacteroidaceae Infections / Q000175:diagnosis* / Q000382:microbiology; D015415:Biomarkers / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000080867:Gingipain Cysteine Endopeptidases; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D016966:Porphyromonas gingivalis / Q000201:enzymology / Q000302:isolation & purification*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012463:Saliva / Q000382:microbiology; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D015415:Biomarkers; D000080867:Gingipain Cysteine Endopeptidases; D019151:Peptide Library; D020033:Protein Isoforms; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D003546:Cysteine Endopeptidases