Multiplexed Protein Biochip Assays--Signal Amplification

描述(申请人提供)：技术创新改变了我们全面分析基因信息的能力。DNA生物芯片和相关技术现在可以同时测量基本上所有人类基因的结构和活动。这一综合能力让我们第一次瞥见了定义新陈代谢和疾病发病机制的潜在分子事件的复杂性。为了补充这一信息，并将其发现转化为人类疾病的诊断和治疗，这些观察结果必须通过直接测量由该遗传信息编码的蛋白质来翻译和扩展。在制药、生物技术和研究界，目前对多路蛋白质检测和量化技术的需求很大，尚未得到满足。目前的多重蛋白质图谱技术在很大程度上依赖于微型化的夹心-ELISA法的应用。然而，这些技术面临着有限的多路复用能力和不同的性能，包括特异性、敏感性和准确性。本提案中描述的创新旨在通过消除在微阵列分析中使用夹心ELISA法的需要并引入信号放大机制来提高分析性能，从而缓解这些限制。这项创新被称为蛋白质足迹扫描技术，它建立在一种新的免疫化学检测方法的基础上，该方法将抗体的特异性与区域特异性氨基酸交联化学相结合，以产生分析物特异性定量。这项技术的成功验证和实施将促进每个目标分析物只使用一种抗体的高灵敏度蛋白质微阵列分析的发展。这反过来将使微阵列具有更高的内容多样性、多样性和新颖性。扩大的内容将对癌症研究特别重要，因为肿瘤发生的多因素性质可能需要并行检查细胞和细胞外蛋白质组室中的大量蛋白质。作为概念验证，将使用由10个经过验证的癌症生物标志物组成的多路蛋白质微阵列分析来验证将这项创新技术应用于蛋白质检测和定量的可行性。

DESCRIPTION (provided by applicant): Technical innovation has transformed our ability to analyze genetic information in a comprehensive fashion. DNA biochips and related technologies now permit the simultaneous measurement of the structures and activities of essentially all human genes. This comprehensive capability has given us our first glimpse of the complexity of the underlying molecular events that define metabolism and disease pathogenesis. To complement this information and translate its findings to the diagnosis and treatment of human disease, these observations must be translated and extended by direct measurement of the proteins that are encoded by this genetic information. Within the pharmaceutical, biotechnology, and research communities, there is currently a large unmet need for multiplexed protein detection and quantification technologies. The current techniques for multiplexed protein profiling rely heavily on application of sandwich-ELISA format in miniaturized scale. However, these techniques suffer limited multiplexing capabilities and variable performance including specificity, sensitivity, and accuracy. The innovation described in this proposal is designed to alleviate these limitations by eliminating the need for the use of sandwich-ELISA in microarray assays and introducing signal amplification mechanism to improve the assay performance. This innovation, termed the protein footprint scanning technology, is founded on a novel immunochemical detection method which combines the specificity of antibodies with regiospecific amino acid cross-linking chemistry to produce analyte-specific quantification. The successful validation and implementation of this technology will catalyze the development of highly sensitive protein microarray assays using only one antibody per target analyte. This will in turn enable microarrays with higher content multiplexity, diversity, and novelty. The expanded content will be especially important for cancer research since the multi-factorial nature of oncogenesis will likely require parallel examination of large numbers of proteins in cellular and extracellular proteome compartments. As a proof-of-concept, the feasibility of applying this innovative technology for protein detection and quantification will be validated using multiplexed protein microarray assays consisting of 10 validated cancer biomarkers.