An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva

Peter Durand Skottrup*, Rodrigo López, Miroslaw Ksiazek, Peter Højrup, Vibeke Baelum, Jan Potempa, Jakub Zbigniew Kaczmarek

*Kontaktforfatter for dette arbejde

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p =.0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499–2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210–1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.

OriginalsprogEngelsk
TidsskriftJournal of Immunological Methods
Vol/bind469
Sider (fra-til)26-32
ISSN0022-1759
DOI
StatusUdgivet - jun. 2019

Fingeraftryk

Saliva
Immunoassay
Periodontitis
Point-of-Care Systems
Surface Plasmon Resonance
Metalloproteases
Periodontal Diseases
Matrix Metalloproteinases
IgY
Enzymes
Pharmaceutical Preparations
Proteins

Citer dette

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title = "An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva",
abstract = "Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10{\%}). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p =.0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499–2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210–1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.",
keywords = "Biomarker, IgY, Inflammation, Karilysin, Periodontitis, Saliva, Virulence factor",
author = "Skottrup, {Peter Durand} and Rodrigo L{\'o}pez and Miroslaw Ksiazek and Peter H{\o}jrup and Vibeke Baelum and Jan Potempa and Kaczmarek, {Jakub Zbigniew}",
year = "2019",
month = "6",
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pages = "26--32",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
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An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva. / Skottrup, Peter Durand; López, Rodrigo; Ksiazek, Miroslaw; Højrup, Peter; Baelum, Vibeke; Potempa, Jan; Kaczmarek, Jakub Zbigniew.

I: Journal of Immunological Methods, Bind 469, 06.2019, s. 26-32.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva

AU - Skottrup, Peter Durand

AU - López, Rodrigo

AU - Ksiazek, Miroslaw

AU - Højrup, Peter

AU - Baelum, Vibeke

AU - Potempa, Jan

AU - Kaczmarek, Jakub Zbigniew

PY - 2019/6

Y1 - 2019/6

N2 - Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p =.0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499–2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210–1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.

AB - Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p =.0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499–2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210–1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.

KW - Biomarker

KW - IgY

KW - Inflammation

KW - Karilysin

KW - Periodontitis

KW - Saliva

KW - Virulence factor

U2 - 10.1016/j.jim.2019.03.003

DO - 10.1016/j.jim.2019.03.003

M3 - Journal article

VL - 469

SP - 26

EP - 32

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

ER -