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

基本信息

  • 批准号:
    9185244
  • 负责人:
  • 金额:
    $ 22.44万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目摘要/摘要 准确和及时地检测循环中的胰岛自身抗体对这两项研究都是至关重要的。 以及1型糖尿病(T1D)患者的临床护理。然而,这一衡量标准在以下方面仍然具有很大的变异性 商业上可用的分析,这种分析也可能不能充分检测出特别低的但与临床相关的 循环中的自身抗体水平。这一逆差转化为错失了及时启动 最适当的治疗方案,并支持亟需的新的和改进的疾病改良研究 干预措施。此外,T1D的大规模公共健康筛查工作因吞吐量低而受阻 当前生物检测的性质,和/或它们对昂贵和专门仪器的要求。我们已经开发出 一项正在申请专利的基于聚合酶链式反应的技术,称为凝集-聚合酶链式反应(ADAP)检测抗体。ADAP 是一种高通量的检测方法,可用于同时检测多种抗体,而只需要非常简单的 少量患者血清(2μL)。ADAP还可以检测自身抗体,灵敏度提高1000到10,000倍 比目前使用的免疫测定法和放射免疫测定法更好,并且可以很容易地集成到公共定量 使用许多医院、诊所和公共卫生机构提供的现有仪器的聚合酶链式反应(QPCR)工作流程 放映地点。重要的是,ADAP代表着与效率较低的PCR驱动的平台的重大背离,例如 免疫-聚合酶链式反应,克服了这类分析固有的许多缺陷,以提供高通量,超灵敏, 稳健、可靠、特异的检测方法。 在这个第一阶段的应用中,我们建议开发一种基于ADAP的检测试剂盒来检测这四种自身抗体 这构成了T1D诊断的基础。相应的GAD65、IA-2、胰岛素和ZnT8抗原将由条形码 与独特的单链DNA序列的接合。抗原与其同系物孵育时的凝集作用 抗体使DNA序列相互接近。然后提供适当的“桥接寡头”,一旦 连接,提供了一个可扩增的DNA双链。这种抗原-自身抗体结合事件提供了一种聚合酶链式反应扩增子 以最小的背景信号实现超灵敏的检测。接下来,我们将使用这些经过适当验证的试剂 用于在诊断前和诊断后分析T1D背景下的患者血清样本。最后,它越来越多地 认识到高亲和力自身抗体是疾病严重程度的特权指标。在对诊断结果的预期中 这一新发现的价值,我们还建议创建一种创新的ADAP分析变体,我们称之为μADAP 实现高亲和力自身抗体的高通量量化。部署这种化验方法可能会允许更多 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 平台,但在敏感度、速度、可重复性和 可经济高效地集成到现有实验室和临床工作流程中的多功能性。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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:
  • 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
  • 通讯作者:
    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:
  • 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
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David Seftel其他文献

David Seftel的其他文献

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{{ truncateString('David Seftel', 18)}}的其他基金

Development of An Automated High-Throughput Dried Blood Spot Assay to Facilitate Large Scale Screening for Type 1 Diabetes Risk
开发自动化高通量干血斑测定法以促进大规模筛查 1 型糖尿病风险
  • 批准号:
    10020787
  • 财政年份:
    2019
  • 资助金额:
    $ 22.44万
  • 项目类别:
Development of An Automated High-Throughput Dried Blood Spot Assay to Facilitate Large Scale Screening for Type 1 Diabetes Risk
开发自动化高通量干血斑测定法以促进大规模筛查 1 型糖尿病风险
  • 批准号:
    9910015
  • 财政年份:
    2019
  • 资助金额:
    $ 22.44万
  • 项目类别:
Multiplex food allergy diagnostic based on IgE immunologic markers
基于 IgE 免疫标记物的多重食物过敏诊断
  • 批准号:
    10011621
  • 财政年份:
    2018
  • 资助金额:
    $ 22.44万
  • 项目类别:
Advancing an accessible, high-throughput and multiplex islet autoantibody test with self-collected capillary dried blood spots for regulatory clearance
利用自我收集的毛细血管干血点推进易于使用、高通量和多重胰岛自身抗体测试,以供监管审批
  • 批准号:
    10699934
  • 财政年份:
    2016
  • 资助金额:
    $ 22.44万
  • 项目类别:
An Ultrasensitive, High-Throughput, and Low-Cost Autoimmunity Panel for the Early Diagnosis and Improved Treatment of Type 1 Diabetes
用于 1 型糖尿病早期诊断和改进治疗的超灵敏、高通量和低成本自身免疫组合
  • 批准号:
    9559575
  • 财政年份:
    2016
  • 资助金额:
    $ 22.44万
  • 项目类别:

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