Department of Clinical Biochemistry, Copenhagen University Hospital, Hvidovre DK-2650, Hvidovre, Denmark; Novo Nordisk A/S, Global Research Technologies, Research Bioanalysis, Novo Nordisk Park, DK-2760 Måløv, Denmark.
Section of Periodontology, HEALTH, Aarhus University, DK-8000 Aarhus, Denmark.
J Immunol Methods. 2019 Jun;469:26-32. doi: 10.1016/j.jim.2019.03.003. Epub 2019 Mar 14.
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.
福赛拟杆菌是一种革兰氏阴性厌氧菌，与破坏性牙周病的发展有关。福赛拟杆菌分泌金属蛋白酶样酶卡拉利辛。体外系统研究表明，卡拉利辛通过降解补体系统蛋白和通过蛋白水解切割使抗菌肽 LL-37 失活，从而调节宿主免疫反应。这使得卡拉利辛成为药物开发和诊断研究中非常有趣的毒力因子。然而，由于缺乏特异性探针，迄今为止尚未在临床样本中证明卡拉利辛的存在。在本工作中，开发了针对卡拉利辛的高滴度和稳定的亲和纯化禽 IgY 抗体。通过表面等离子体共振成像发现 IgY 的亲和力在纳摩尔范围内。该抗体可通过免疫印迹检测唾液样本中的卡拉利辛，并且在测试人 MMP-3 时具有特异性。此外，开发了基于禽 IgY 的免疫测定法，该方法显示出低的日内和日间测定变异性（CV 低于 10％）。将该免疫测定法应用于具有或不具有牙周炎迹象的青少年的一组特征良好的唾液样本中，表明可以在唾液中检测到卡拉利辛。在具有牙周炎迹象的参与者的唾液与健康对照者的唾液之间，发现卡拉利辛浓度存在显著差异（p=0.0024）。牙周炎病例的卡拉利辛水平中位数为 957pg/ml（IQR，499-2132pg/ml），对照组的中位数为 569pg/ml（IQR，210-1343pg/ml）。总体而言，我们的数据证实了卡拉利辛在临床样本中的存在。所描述的基于 IgY 的免疫测定法可作为临床或即时护理环境中基于蛋白质的生物标志物筛选的一部分证明是有用的。
