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

Peter Durand Skottrup; Paul Leonard; Jakub Kaczmarek; Florian Veillard; Jan Johannes Enghild; Richard O'Kennedy; Aneta Sroka; Rasmus Prætorius Clausen; Jan Potempa; Erik Skjold Riise

doi:10.1016/j.ab.2011.04.015

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

Peter Durand Skottrup, Paul Leonard, Jakub Kaczmarek, Florian Veillard, Jan Johannes Enghild, Richard O'Kennedy, Aneta Sroka, Rasmus Prætorius Clausen, Jan Potempa, Erik Skjold Riise

29 Citations (Scopus)

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.

Original language	English
Journal	Analytical Biochemistry
Volume	415
Issue number	2
Pages (from-to)	158-167
ISSN	0003-2697
DOIs	https://doi.org/10.1016/j.ab.2011.04.015
Publication status	Published - 15 Aug 2011

Keywords

Former Faculty of Pharmaceutical Sciences

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10.1016/j.ab.2011.04.015

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@article{c3976f3a8b2b4a60b622009591f1a6bd,

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.",

keywords = "Former Faculty of Pharmaceutical Sciences",

author = "Skottrup, {Peter Durand} and Paul Leonard and Jakub Kaczmarek and Florian Veillard and {Johannes Enghild}, Jan and Richard O'Kennedy and Aneta Sroka and Clausen, {Rasmus Pr{\ae}torius} and Jan Potempa and Riise, {Erik Skjold}",

year = "2011",

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doi = "10.1016/j.ab.2011.04.015",

language = "English",

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journal = "Analytical Biochemistry",

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T1 - Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

AU - Skottrup, Peter Durand

AU - Leonard, Paul

AU - Kaczmarek, Jakub

AU - Veillard, Florian

AU - Johannes Enghild, Jan

AU - O'Kennedy, Richard

AU - Sroka, Aneta

AU - Clausen, Rasmus Prætorius

AU - Potempa, Jan

AU - Riise, Erik Skjold

PY - 2011/8/15

Y1 - 2011/8/15

N2 - 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.

AB - 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.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1016/j.ab.2011.04.015

DO - 10.1016/j.ab.2011.04.015

M3 - Journal article

SN - 0003-2697

VL - 415

SP - 158

EP - 167

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

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