Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).
Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).
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DOI：
10.1021/acscentsci.5b00340
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发表时间：
2016-03-23
影响因子：
18.2
通讯作者：
Bertozzi CR
中科院分区：
文献类型：
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作者：
Tsai CT;Robinson PV;Spencer CA;Bertozzi CR
抗体是许多疾病诊断中广泛使用的生物标志物。基于固相固定抗原的检测方法构成了大多数临床抗体检测平台，但会受到抗原变性和表位掩蔽的影响。这些技术障碍在检测与非线性或构象表位结合的抗体时尤其麻烦，例如1型糖尿病患者的抗胰岛素抗体和与甲状腺癌相关的抗甲状腺球蛋白抗体。放射免疫分析仍然是这些具有挑战性的抗体生物标记物的黄金标准，但有限的多重性和对危险放射性试剂的依赖使它们无法在专门的检测设施之外使用。在这里，我们提出了一种超灵敏的检测抗体的溶液相方法，称为凝集-聚合酶链式反应(ADAP)检测抗体。抗体结合并凝集合成的抗原-DNA偶联物，使DNA链连接并随后通过qPCR进行定量。ADAP检测L样品中2μ抗体的Zepto-to分子，动态范围为5-6个数量级。使用ADAP，我们从患者血浆中检测到抗甲状腺球蛋白自身抗体，其灵敏度比FDA批准的放射免疫分析方法提高了1000倍。最后，我们通过在一次实验中同时检测多个抗体来证明ADAP的多功能性。ADAP结合了简单性、敏感性、广泛的动态范围、多重性和使用标准的PCR协议，为发现和检测抗体生物标记物创造了新的机会。我们已经建立了一种利用抗原−DNA结合物超灵敏地检测抗体的定量聚合酶链式反应方法。抗体的多价性和凝集倾向推动了邻近效应，以促进连接时DNA扩增的产生。
Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers. We have developed a qPCR assay to ultrasensitively detect antibodies using antigen−DNA conjugates. The multivalency and agglutination propensity of antibodies drive a proximity effect to facilitate generation of DNA amplicons upon ligation.