Multiplexed Protein Biochip Assays--Signal Amplification

多重蛋白质生物芯片检测——信号放大

• 批准号: 6964910
• 负责人: Raymond Kim
• 金额: $ 9.98万
• 依托单位: GENEPRISM, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6964910
• 关键词: bioassay; biomarker; biotechnology; diagnosis quality /standard; method development; microarray technology; neoplasm /cancer diagnosis; protein quantitation /detection; proteomics

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.

技术创新已经改变了我们以全面的方式分析遗传信息的能力。 DNA生物芯片和相关技术现在允许同时测量基本上所有人类基因的结构和活性。 这种全面的能力使我们第一次看到了定义代谢和疾病发病机制的潜在分子事件的复杂性。 为了补充这些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这些遗传信息编码的蛋白质来进行翻译和扩展。 在制药、生物技术和研究领域，目前对多重蛋白质检测和定量技术的需求很大。 目前用于多重蛋白质谱分析的技术严重依赖于微型化规模的ELISA形式的应用。然而，这些技术遭受有限的多路复用能力和可变的性能，包括特异性、灵敏度和准确性。 本提案中描述的创新旨在通过消除在微阵列测定中使用ELISA的需要并引入信号放大机制来改善测定性能来减轻这些限制。 这项创新被称为蛋白质足迹扫描技术，是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高度敏感的蛋白质微阵列检测的发展，每个目标分析物仅使用一种抗体。 这将反过来使微阵列具有更高的内容复用性、多样性和新奇。 扩大的内容将是特别重要的癌症研究，因为肿瘤发生的多因素性质可能需要大量的蛋白质在细胞和细胞外蛋白质组室的平行检查。 作为概念验证，将使用由10种经验证的癌症生物标志物组成的多重蛋白质微阵列测定来验证应用这种创新技术进行蛋白质检测和定量的可行性。