Antibody-Detection-by-Agglutination-PCR (ADAP): An Ultra-Sensitive, High-Throughput, Multiplexable Tool for T1D Diagnosis and Monitoring

凝集 PCR 抗体检测 (ADAP)：一种用于 T1D 诊断和监测的超灵敏、高通量、可多重工具

• 批准号: 9185244
• 负责人: David Seftel
• 金额: $ 22.44万
• 依托单位: ENABLE BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185244
• 关键词: Affect; Affinity; Agglutination; Antibodies; Antigens; Autoantibodies; Autoimmune Diseases; Binding; Biological Assay; Child; Clinical; Clinics and Hospitals; DNA; DNA Sequence; Data; Detection; Development; Diabetes Mellitus; Diabetes autoantibodies; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diagnostic Specificity; Disease; Disease Management; Early Diagnosis; Effectiveness; Equipment; Evaluation; Event; First Degree Relative; Gold; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Intervention; Islets of Langerhans; Laboratories; Legal patent; Life; Ligation; Link; Measurement; Methods; Monitor; Nature; Newly Diagnosed; Oligonucleotides; Onset of illness; Outcome; Patients; Phase; Plasma; Preparation; Public Health; Radio; Reagent; Reproducibility; Research; Sampling; Serum; Severity of illness; Signal Transduction; Single-Stranded DNA; Site; Solid; Source; Speed; Statistical Data Interpretation; Statistical Models; Technology; Testing; Therapeutic Intervention; Time; Translating; Treatment Protocols; Validation; Variant; Vendor; base; clinical care; clinically relevant; cost; head-to-head comparison; high throughput screening; improved; innovation; instrumentation; multiplex detection; neonate; novel; outcome forecast; prognostic value; screening; tool

PROJECT SUMMARY/ABSTRACT Accurate and timely detection of circulating autoantibodies against pancreatic islet antigens is critical to both research and clinical care for patients with type 1 diabetes (T1D). However this measurement remains highly variable across commercially available assays, and such assays also may not adequately detect particularly low but clinically relevant levels of circulating autoantibodies. This deficit translates into missed opportunities for both the timely initiation of the most appropriate treatment regimens and to support much needed research into novel and improved disease-modifying interventions. Additionally, large-scale public health screening efforts for T1D are hampered by the low throughput nature of current bioassays, and/or their requirement for expensive and specialized instrumentation. We have developed a proprietary patent-pending PCR-based technology termed Antibody Detection by Agglutination-PCR (ADAP). ADAP is a high-throughput assay that can be used for simultaneous detection of multiple antibodies while requiring only a very small amount of patient serum (2 μL). ADAP also detects autoantibodies with 1,000 to 10,000 times greater sensitivity than the currently used immunometric and radio-immuno assays, and can be readily integrated into common quantitative PCR (qPCR) workflows using pre-existing instrumentation that is available at many hospitals, clinics and public health screening sites. Importantly, ADAP represents a significant departure from less effective PCR-driven platforms such as immuno-PCR, overcoming many of the deficits inherent to this class of assays to afford a high-throughput, ultrasensitive, robust, reliable, and specific detection method. In this Phase 1 application, we propose to develop an ADAP-based assay kit for detection of the four autoantibodies that form the basis for T1D diagnosis. The corresponding GAD65, IA-2, insulin, and ZnT8 antigens will be barcoded by conjugation to unique single-stranded DNA sequences. Agglutination of an antigen upon incubation with its cognate antibody brings the DNA sequences near to each other. An appropriate “bridge oligo” is then supplied which, upon ligation, affords an amplifiable DNA duplex. This antigen-autoantibody binding event provides a PCR amplicon that enables ultrasensitive detection with minimal background signal. Next we will use these appropriately validated reagents for analysis of patient serum samples in the context of T1D, both before and after diagnosis. Finally, it is increasingly appreciated that high-affinity autoantibodies are privileged indicators of disease severity. In anticipation of the diagnostic value of this new finding, we also propose to create an innovative variant of the ADAP assay which we term μADAP to enable high throughput quantification of high-affinity autoantibodies. Deployment of such an assay may allow more accurate determination of T1D prognosis, and enable improved choices of therapeutic interventions. In summary, we seek to establish the ADAP T1D assay kit not only as an effective alternative to current T1D diagnostic platforms, but one with significantly expanded capabilities in terms of sensitivity, speed, reproducibility and multiplexability that can be cost-effectively integrated into existing laboratory and clinical workflows.

项目概要/摘要 准确及时地检测针对胰岛抗原的循环自身抗体对于这两项研究都至关重要 以及 1 型糖尿病 (T1D) 患者的临床护理。然而，这种测量在不同地区仍然存在很大差异 商业上可用的测定，并且此类测定也可能无法充分检测特别低但临床相关的 循环自身抗体的水平。这种赤字意味着错失了及时启动 最合适的治疗方案并支持急需的新型和改进的疾病缓解研究 干预措施。此外，针对 T1D 的大规模公共卫生筛查工作因通量低而受到阻碍 当前生物测定的性质，和/或它们对昂贵和专业仪器的要求。我们开发了 一项正在申请专利的基于 PCR 的专有技术，称为凝集 PCR 抗体检测 (ADAP)。阿达普 是一种高通量测定法，可用于同时检测多种抗体，同时只需要非常 少量患者血清（2 μL）。 ADAP 还可以以 1,000 至 10,000 倍的灵敏度检测自身抗体 比目前使用的免疫测定和放射免疫测定更先进，并且可以很容易地集成到常见的定量分析中 PCR (qPCR) 工作流程使用许多医院、诊所和公共卫生部门现有的仪器 筛选地点。重要的是，ADAP 与效率较低的 PCR 驱动平台（例如 免疫 PCR，克服了此类检测固有的许多缺陷，提供了高通量、超灵敏、 稳健、可靠、特异性的检测方法。 在此一期应用中，我们建议开发一种基于 ADAP 的检测试剂盒，用于检测四种自身抗体 构成 T1D 诊断的基础。相应的 GAD65、IA-2、胰岛素和 ZnT8 抗原将通过以下方式进行条形码标记 与独特的单链 DNA 序列缀合。抗原与其同源物孵育后发生凝集 抗体使 DNA 序列彼此靠近。然后提供适当的“桥寡核苷酸”， 连接，提供可扩增的 DNA 双链体。这种抗原-自身抗体结合事件提供了一个 PCR 扩增子， 能够以最小的背景信号实现超灵敏检测。接下来我们将使用这些经过适当验证的试剂 用于在诊断前后分析 T1D 患者血清样本。最后，越来越多 认识到高亲和力自身抗体是疾病严重程度的重要指标。期待诊断结果 鉴于这一新发现的价值，我们还建议创建 ADAP 检测的创新变体，我们将其命名为 μADAP 实现高亲和力自身抗体的高通量定量。这种检测的部署可能会允许更多 准确确定 T1D 预后，并改进治疗干预措施的选择。 总之，我们寻求建立 ADAP T1D 检测试剂盒，不仅作为当前 T1D 诊断的有效替代方案 平台，但在灵敏度、速度、再现性和 多重性，可以经济高效地集成到现有实验室和临床工作流程中。

项目成果

Automation of a multiplex agglutination-PCR (ADAP) type 1 diabetes (T1D) assay for the rapid analysis of islet autoantibodies.

• DOI: 10.1016/j.slast.2021.10.001
• 发表时间: 2021-10
• 期刊: SLAS technology
• 影响因子: 2.7
• 作者: Felipe de Jesus Cortez;David Gebhart;Devangkumar Tandel;Peter V. Robinson;D. Seftel;Darrell M. Wilson;D. Maahs;B. Buckingham;K. Miller;Cheng-ting Tsai

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

• DOI: 10.1371/journal.pone.0242049
• 发表时间: 2020
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Cortez FJ;Gebhart D;Robinson PV;Seftel D;Pourmandi N;Owyoung J;Bertozzi CR;Wilson DM;Maahs DM;Buckingham BA;Mills JR;Roforth MM;Pittock SJ;McKeon A;Page K;Wolf WA;Sanda S;Speake C;Greenbaum CJ;Tsai CT
• 通讯作者: Tsai CT