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

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Harvard

Skottrup, PD, Leonard, P, Kaczmarek, JZ, Veillard, F, Enghild, JJ, O'Kennedy, R, Sroka, A, Clausen, RP, Potempa, J & Riise, E 2011, 'Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis', Analytical Biochemistry, bind 415, nr. 2, s. 158-67. https://doi.org/10.1016/j.ab.2011.04.015

APA

Skottrup, P. D., Leonard, P., Kaczmarek, J. Z., Veillard, F., Enghild, J. J., O'Kennedy, R., Sroka, A., Clausen, R. P., Potempa, J., & Riise, E. (2011). Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis. Analytical Biochemistry, 415(2), 158-67. https://doi.org/10.1016/j.ab.2011.04.015

CBE

Skottrup PD, Leonard P, Kaczmarek JZ, Veillard F, Enghild JJ, O'Kennedy R, Sroka A, Clausen RP, Potempa J, Riise E. 2011. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis. Analytical Biochemistry. 415(2):158-67. https://doi.org/10.1016/j.ab.2011.04.015

MLA

Vancouver

Author

Skottrup, Peter Durand ; Leonard, Paul ; Kaczmarek, Jakub Zbigniew ; Veillard, Florian ; Enghild, Jan J. ; O'Kennedy, Richard ; Sroka, Aneta ; Clausen, Rasmus Prætorius ; Potempa, Jan ; Riise, Erik. / Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis. I: Analytical Biochemistry. 2011 ; Bind 415, Nr. 2. s. 158-67.

Bibtex

@article{8fdb54e8d6104d3bb20f2c550db7daae,
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 = "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",
author = "Skottrup, {Peter Durand} and Paul Leonard and Kaczmarek, {Jakub Zbigniew} and Florian Veillard and Enghild, {Jan J.} and Richard O'Kennedy and Aneta Sroka and Clausen, {Rasmus Pr{\ae}torius} and Jan Potempa and Erik Riise",
note = "Copyright {\textcopyright} 2011 Elsevier Inc. All rights reserved.",
year = "2011",
month = aug,
day = "15",
doi = "10.1016/j.ab.2011.04.015",
language = "English",
volume = "415",
pages = "158--67",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press",
number = "2",

}

RIS

TY - JOUR

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 Zbigniew

AU - Veillard, Florian

AU - Enghild, Jan J.

AU - O'Kennedy, Richard

AU - Sroka, Aneta

AU - Clausen, Rasmus Prætorius

AU - Potempa, Jan

AU - Riise, Erik

N1 - Copyright © 2011 Elsevier Inc. All rights reserved.

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 - Adhesins, Bacterial

KW - Amino Acid Sequence

KW - Antibodies, Bacterial

KW - Bacteroidaceae Infections

KW - Biological Markers

KW - Cysteine Endopeptidases

KW - Humans

KW - Molecular Sequence Data

KW - Peptide Library

KW - Porphyromonas gingivalis

KW - Protein Binding

KW - Protein Isoforms

KW - Recombinant Proteins

KW - Saliva

KW - Single-Chain Antibodies

U2 - 10.1016/j.ab.2011.04.015

DO - 10.1016/j.ab.2011.04.015

M3 - Journal article

C2 - 21569755

VL - 415

SP - 158

EP - 167

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -

ID: 44863385