Mulitplexed protein measurement by real-time immuno-PCR

描述(申请人提供)：实时聚合酶链式反应(PCR)是定量基因表达谱最常用的方法；它允许同时对数百个mRNA转录本进行极其灵敏的测量。我们提出了一种方法，可以用同样的技术来定量测定蛋白质的丰度。这是通过实施夹心免疫分析格式实现的，在该格式中，用不同的DNA序列标记检测抗体。然后使用实时荧光聚合酶链式反应对所有DNA标记进行并行量化，从而揭示生物样本中同源蛋白质的数量。初步实验表明，使用该抗体-DNA偶联物的实时定量聚合酶链式反应的灵敏度比标准ELISA提高了两个数量级以上，动态范围增加了近三个数量级，标准偏差很低(变异系数约为5%)。还获得了支持这种方法多重能力的初步证据。

DESCRIPTION (provided by applicant): The real-time polymerase chain reaction (PCR) is the most commonly used method for quantitative gene expression profiling; it allows extremely sensitive measurement of hundreds of mRNA transcripts in parallel. We propose a method by which protein abundances can be quantitated using the same technology. This is achieved by implementing a sandwich immuno-assay format in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary experiments show that real-time PCR using such antibody-DNA conjugates gives over two orders of magnitude more sensitive detection and almost three orders of magnitude increased dynamic range in comparison to standard ELISA, and standard deviations are very low (CV approximately 5%). Initial evidence supporting the ability to multiplex this method has also been obtained.