Development of An Automated High-Throughput Dried Blood Spot Assay to Facilitate Large Scale Screening for Type 1 Diabetes Risk

1型糖尿病（T1 D）是一种慢性自身免疫性疾病，影响世界上数百万儿童和成人。 我们高达40%的T1 D患者在急诊室被诊断为患有危及生命的糖尿病 酮症酸中毒（DKA），导致严重的临床并发症和巨大的经济负担。 筛查早期T1 D（抗胰岛细胞自身抗体）的血源性标志物的程序可以 显著降低DKA的发生率，改善患者的生活质量。然而，大规模 这种筛选活动的实施受到高成本和低通量的阻碍， 放射免疫测定法（RIA），测量抗胰岛自身抗体的金标准方法。而且 筛查项目成本的很大一部分与静脉切开术、冷链储存和 送货.为了解决这一问题，Enable Biosciences的目标是开发一种具有成本效益的，多元化的，高 通过通量和非放射性免疫测定诊断T1 D并筛查T1 D风险个体 使用低成本的易于收集的干血点（DBS）。这一高科技的成功发展， 通过量干血斑T1 D测定可以显著增加一般人群的有效性- T1 D筛查项目。在JDRF和斯坦福大学支持的一项倡议中，我们感到满意的是， 基于血清的Enable检测试剂盒实现了抗GAD、抗IA 2的最高性能， 在最近的2018年设盲胰岛细胞试验中，抗胰岛素抗体的性能仅次于最高 自身抗体标准化计划（IASP）的竞争出50个参与实验室。 IASP数据是使用定制的Enable试剂盒，通过自动化的Enable血清测定生成的。 这是一个与汉密尔顿机器人公司合作开发的分析仪。此外，我们证明了我们的手动DBS- 基于Enable的测定与基于血清的测定强烈相关（R=0.90-0.96）。一致性作为 通过Cohen检验评估，显示kappa在0.85-0.89之间。积极和消极的协议， 所有三种上述自身抗体的范围为85%-99%。值得注意的是，我们还展示了不到 当DBS卡在37°C下储存4周时，测定信号降解10%。根据这些 坚实的技术成果，我们认为这是我们的“第一阶段样的结果”，详细描述在这一点上， 建议，我们的目标是进一步自动化我们的DBS测定程序的高通量功能 包括DBS洗脱和使用不同样品的DBS洗脱液测试，例如来自斯坦福大学的样品 大学医学院。我们希望提供一个集成的DBS T1 D自身抗体检测 （洗脱/检测自动化设备和试剂盒），以服务于大型筛查中心和CLIA- laboratories.我们进一步计划获得CE标志分类和FDA批准，以进一步分散 DBS T1 D自身抗体检测能够满足社区、临床医生和 全世界的研究人员。

Type 1 diabetes (T1D) is a chronic autoimmune disease that affects millions of children and adults in the US. Up to 40% of T1D patients are diagnosed in the emergency room with life-threatening diabetic ketoacidosis (DKA), resulting in severe clinical complications and a substantial economic burden. Programs that screen for blood-borne markers of early-stage T1D (anti-islet cell autoantibodies) can significantly diminish the incidence of DKA and improve patient quality of life. However, large-scale implementation of such screening campaigns is hindered by the high cost and low throughput of radioimmunoassay (RIA), the gold-standard method for measuring anti-islet autoantibodies. Moreover, a significant portion of screening program costs are associated with phlebotomy, cold-chain storage and shipping. To address this, Enable Biosciences aims to develop a cost-effective, multiplex, high- throughput and non-radioactive immunoassay to diagnose T1D and screen for individuals at risk for T1D using low-cost easy-to-collect dried blood spots (DBS). The successful development of this high- throughput dried blood spot T1D assay can substantially increase the effectiveness of general population- based T1D screening programs. In a JDRF- and Stanford-supported initiative, we were gratified that a serum-based Enable assay achieved the highest performance for anti-GAD, anti-IA2 and a close second-to-highest performance for anti-insulin antibodies in the recent blinded 2018 Islet-cell Autoantibody Standardization Program (IASP) competition out of 50 participating laboratories. The IASP data were generated with an automated Enable serum assay using a custom-made Enable analyzer created in collaboration with Hamilton Robotics. Furthermore, we showed that our manual DBS- based Enable assay correlated strongly with a serum-based assay (R=0.90-0.96). The concordance as evaluated by Cohen’s test showed kappa between 0.85-0.89. The positive and negative agreements for all three aforementioned autoantibodies range from 85%-99%. Notably, we also demonstrated less than 10% degradation of assay signals when DBS cards were stored at 37°C for 4 weeks. Building on these solid technical achievements, which we consider as our “Phase I-like Results” described in detail in this proposal, we aim to further automate our DBS assay procedures for high-throughput functionality including DBS elution and DBS eluent testing using diverse samples, such as those from Stanford University School of Medicine. We expect to deliver an integrated DBS T1D autoantibody assay (elution/testing automation device and reagent kits) to serve large screening centers and CLIA- laboratories. We further plan to obtain CE Mark classification and FDA approval to further decentralize DBS T1D autoantibody testing to be able to address the important needs of communities, clinicians and researchers worldwide.