Department of Anthropology, University of Oregon, Eugene, OR, 97403, USA.
Department of Anthropology, University of Colorado Springs, Colorado Springs, CO, 80918, USA.
J Physiol Anthropol. 2020 Jul 16;39(1):16. doi: 10.1186/s40101-020-00228-8.
The prevalence of allergic and autoimmune conditions has been steadily increasing in wealthy nations over the past century. One hypothesis put forward to explain this is the Old Friends Hypothesis, which posits that increased hygiene, urbanization, and lifestyle changes have reduced our exposure to parasites and microbes that we co-evolved with, resulting in immune dysregulation. However, research in traditionally living populations, who are exposed to greater parasite and pathogen loads such as those encountered during our evolution, is limited, in part due to a lack of minimally invasive, field-friendly biomarkers of autoimmune disorders. We therefore developed an ELISA to assess positivity for thyroid peroxidase autoantibody (TPO-Ab), an indicator of autoimmune thyroid disease, based on dried blood spot (DBS) samples.
We used the Accubind anti-thyroid peroxidase test system to screen our validation samples comprising matched fingerprick DBS, venous DBS, and plasma samples from 182 adults. After confirming that we had TPO-Ab-positive individuals in our validation sample (n = 12), we developed an indirect ELISA to measure TPO-Ab levels from one 3-mm DBS punch. The sensitivity and specificity of our assay for DBS samples ranged from 91.7-100% and 98.2-98.8%, respectively, using a cut-off value of ≥ 26 IU/mL. Intra-assay reliability for duplicate quality control DBS punches was 5.2%, while inter-assay reliability ranged from 11.5-24.4% for high, medium, and low DBS controls. Dilutional linearity ranged from 80 to 120%, and spike and recovery experiments indicated that the DBS matrix does not interfere with the detection of TPO-Ab. TPO-Ab levels remained stable in DBS samples stored at - 28 °C or - 80 °C, but decreased over time in DBS samples kept at 22 °C or at 37 °C.
We developed an in-house, kit-independent indirect ELISA assay to determine individuals' TPO-Ab positivity based on dried blood spots, representing a cost-effective method with potential applications in a range of research settings.
在过去的一个世纪里，富裕国家中过敏和自身免疫性疾病的患病率一直在稳步上升。有一种假说解释了这一现象，即“老朋友假说”，该假说认为，卫生条件改善、城市化和生活方式的改变减少了我们与寄生虫和微生物的接触，而这些寄生虫和微生物是我们共同进化而来的，导致免疫失调。然而，在传统生活人群中的研究受到限制，这些人群接触到更多的寄生虫和病原体，例如在我们进化过程中遇到的那些，部分原因是缺乏微创、适合野外的自身免疫性疾病生物标志物。因此，我们开发了一种酶联免疫吸附试验（ELISA），以评估甲状腺过氧化物酶自身抗体（TPO-Ab）的阳性率，这是自身免疫性甲状腺疾病的一个指标，基于干血斑（DBS）样本。
我们使用 Accubind 抗甲状腺过氧化物酶检测系统筛选了我们的验证样本，这些样本包括来自 182 名成年人的匹配指血 DBS、静脉 DBS 和血浆样本。在确认我们的验证样本中有 TPO-Ab 阳性个体（n=12）后，我们开发了一种间接 ELISA 来测量来自一个 3 毫米 DBS 打孔的 TPO-Ab 水平。我们的 DBS 样本检测方法的灵敏度和特异性分别为 91.7-100%和 98.2-98.8%，使用的截断值为≥26IU/mL。用于重复质量控制 DBS 打孔的室内可靠性为 5.2%，而高、中、低 DBS 对照的室内可靠性范围为 11.5-24.4%。稀释线性范围为 80-120%，而添加和回收实验表明 DBS 基质不干扰 TPO-Ab 的检测。在-28°C 或-80°C 下储存的 DBS 样本中 TPO-Ab 水平保持稳定，但在 22°C 或 37°C 下储存的 DBS 样本中 TPO-Ab 水平随时间推移而降低。
我们开发了一种基于干血斑的内部、试剂盒独立的间接 ELISA 检测方法来确定个体的 TPO-Ab 阳性率，这是一种具有成本效益的方法，具有在一系列研究环境中应用的潜力。
