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

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  • Peter Durand Skottrup, Department of Clinical Biochemistry, Copenhagen University Hospital, Hvidovre DK-2650, Denmark, Novo Nordisk A/S, Global Research Technologies, Research Bioanalysis, Novo Nordisk Park, DK-2760 Måløv, Denmark, Danmark
  • Rodrigo López
  • Miroslaw Ksiazek, Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, Department of Oral Immunology and Infectious Diseases, University of Louisville, School of Dentistry, Louisville, KY, USA, Polen
  • Peter Højrup, Department of Biochemistry and Molecular Biology, University of Southern Denmark , Danmark
  • Vibeke Baelum
  • Jan Potempa, Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, Department of Oral Immunology and Infectious Diseases, University of Louisville, School of Dentistry, Louisville, KY, USA, Polen
  • Jakub Zbigniew Kaczmarek, Department of Biochemistry and Molecular Biology, University of Southern Denmark , Research and Development Department, Sanovo Biotech A/S, DK-5000 Odense, Denmark, Danmark

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
Antal sider7
ISSN0022-1759
DOI
StatusUdgivet - 2019

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Copyright © 2019. Published by Elsevier B.V.

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