Multiplexed Protein Measurement by Real-time Immuno-PCR

描述（由申请人提供）：已经开发了许多用于并行测量多种蛋白质的蛋白质组学平台，但由于它们的灵敏度都低于标准ELISA测定的数量级，因此采用受到限制。相比之下，并行定量测量多个mrna水平的方法非常稳健和敏感。实时聚合酶链反应（real-time polymerase chain reaction， PCR）是最灵敏、最常用的基因表达定量分析方法；它可以同时对数百个mRNA转录物进行极其灵敏的测量。我们现在已经开发了一种专有的方法，用于使用实时pcr技术来测量蛋白质浓度，也是以多路复用的方式，具有非常高的灵敏度和可重复性。该方法，多重实时免疫pcr (MRI-PCR)，包括一个三明治免疫测定，其中检测抗体被标记不同的DMA序列。实时聚合酶链式反应（Real-time PCR）用于同时定量所有DNA标记，从而揭示生物样品中同源蛋白的数量。I期数据显示，与标准的ELISA检测相比，采用MRI-PCR检测的多重蛋白检测灵敏度提高了约2个数量级，动态范围增加了约2个数量级，并且具有非常低的标准偏差（CV <10%）和高特异性。在II期，我们建议开发两个商业准备的12个细胞因子和12个脂肪因子的十二聚体检测组，分别针对炎症和代谢疾病的研究需求。

DESCRIPTION (provided by applicant): Numerous proteomics platforms for measuring multiple proteins in parallel have been developed, but adoption has been limited by the fact that they all are orders of magnitude less sensitive than standard ELISA assays. By contrast, methods for quantitatively measuring the levels of multiple mRNAs in parallel are very robust and sensitive. The real-time polymerase chain reaction (PCR) is the most sensitive and commonly used method for quantitative gene expression profiling; it allows extremely sensitive measurement of hundreds of mRNA transcripts in parallel. We have now developed a proprietary method for using the real-time-PCR technology to measure protein concentrations, also in a multiplexed fashion and with very high sensitivity and reproducibility. The method, Multiplexed Real-time-lmmuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DMA 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. Phase I data show that multiplexed protein measurements employing MRI-PCR assays provides about 2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase II, we propose to develop two commercial-ready panels of dodecaplexed assays for 12 cytokines and 12 adipokines, targeting research needs in inflammatory and metabolic diseases, respectively.