Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, DB21, James Cook Drive, Townsville, QLD 4811, Australia.
Analyst. 2012 Nov 21;137(22):5193-6. doi: 10.1039/c2an35857c. Epub 2012 Sep 21.
Protein detection in complex biological fluids and matrices has become a widely diversified field utilizing a number of different technologies. The quantification of target proteins in complex media such as serum remains a challenge for most technologies such as mass spectrometry, ELISA and western blot. Quantitative Immuno-PCR has been heavily used for antigen detection in immunoassays, but minimally so for quantifying affinity-tagged proteins expressed or circulating in complex matrices--despite its high sensitivity and robustness--because it suffers from detrimental background effects arising from its extreme detection power. We report the development of a universal qIPCR-based platform for the reproducible detection of dual affinity-tagged protein analytes in crude complex matrices such as serum and cell culture media or lysates. The system uses a couple of high-affinity antibodies against two affinity tags (GFP and HA) for the detection of dual-tagged proteins. The dual-tagged analyte is immuno-captured by one of its tags, while the second tag is bound by a detection device consisting of a new kind of self-assembled antibody-DNA conjugate. The new qIPCR platform enabled picomolar quantification of dual-tagged sortase in crude serum in 4 h including the PCR step.
在复杂的生物流体和基质中进行蛋白质检测已经成为一个广泛多样化的领域，利用了许多不同的技术。大多数技术，如质谱、ELISA 和 Western blot，都难以对血清等复杂介质中的目标蛋白质进行定量。定量免疫-PCR 已被广泛用于免疫分析中的抗原检测，但由于其极高的灵敏度和稳健性，对在复杂基质中表达或循环的亲和标记蛋白的定量应用很少，因为它会受到其极端检测能力产生的有害背景效应的影响。我们报告了一种通用的基于 qIPCR 的平台的开发，用于在粗制复杂基质（如血清和细胞培养基或裂解物）中可重复地检测双重亲和标记蛋白分析物。该系统使用针对两种亲和标签（GFP 和 HA）的一对高亲和力抗体来检测双重标记的蛋白质。双重标记的分析物通过其一个标签进行免疫捕获，而第二个标签则与由新型自组装抗体-DNA 偶联物组成的检测设备结合。新的 qIPCR 平台能够在包括 PCR 步骤在内的 4 小时内对粗制血清中的双标签 sortase 进行皮摩尔级定量。
