Colocalization-by-linkage assay on microparticles (CLAMP): A multiplex immunoassay platform for quantitative proteomics

蛋白质是临床上分析的最常用的生物标记物，它们也被用于研究和药物开发。今天，用于蛋白质分析的全球免疫分析市场价值200亿美元。在过去的50年里，金标准的蛋白质检测方法仍然是夹心免疫分析法。夹心免疫分析使用一对抗体，每个抗体对目标蛋白质高度特异，以冗余地识别样本中的蛋白质。虽然人们希望和需要同时可靠和灵敏地测量许多蛋白质，但事实证明，多重夹心免疫分析非常困难。阻碍这一进展的根本问题早已为人所知：要测量多种蛋白质，必须加入多种抗体试剂并将其混合在一起，但这些试剂具有粘性和交叉反应。因此，在这些混合物中，抗体与非目标蛋白发生交叉反应，或相互粘连，从而产生假阳性结合信号和无法从实际目标蛋白结合信号中分离出来的噪音。**我们开发了一种夹心免疫分析平台，通过首先防止不同抗体对的混合，克服了多重分析中的交叉反应和粘连问题。因此，我们的技术可以可靠和灵敏地一次测量样品中的多个蛋白质。在这个I2I阶段1项目中，我们建议将平台从概念验证推进到市场准备就绪的原型。我们建议将同时测量的蛋白质数量增加到50个，并通过测量癌症患者和健康对照的血液中的蛋白质来进行测试。重要的是，我们的方法也是用户友好的，适合生物和临床研究的现有实验工作流程，并通过利用广泛可用的仪器提供了较低的进入门槛。这些功能和我们的结果突显了该平台的巨大商业潜力，这个I2I项目将使该平台成为一种适合投资和进一步商业化的市场就绪技术。我们预计，我们的平台将为学术界和工业界的蛋白质组学研究开辟新的途径，创造新的就业机会，并发展加拿大的免疫分析行业。

Proteins are the most commonly used biomarker analyzed in the clinic, and they are also measured for research and drug development. Today, the global immunoassay market for protein analysis is valued at $20B. For the past 50 years, the gold standard protein detection method has remained the sandwich immunoassay. A sandwich immunoassay uses a pair of antibodies, each highly specific for the target protein, to redundantly identify the protein within a sample. Whereas there is a desire and need to measure many proteins at once reliably and sensitively, it has proven very difficult to multiplex sandwich immunoassays. The underlying problem that prevents progress has long been known: to measure multiple proteins, multiple antibody reagents must be added and mixed together, but these reagents are "sticky" and cross-reactive. Hence, within these mixtures, antibodies cross-react with non-target proteins, or stick to one another, thus creating false positive binding signals and noise that cannot be untangled from the actual target protein-binding signal.** We have developed a sandwich immunoassay platform that overcomes cross-reactivity and sticking problems in multiplexed assays by preventing mixing of different antibody pairs in the first place. Hence, our technology can reliably and sensitively measure multiple proteins in a sample at once. In this I2I Phase 1 project, we propose to advance the platform from a proof-of-concept to a market-ready prototype. We propose to increase the number of proteins measured simultaneously to 50, and to test it by measuring proteins in the blood of cancer patients and healthy controls. Importantly, our approach is also user-friendly, fits within existing experimental workflows in biological and clinical research, and offers a low entry barrier by taking advantage of widely available instrumentation. These features and our results underline a significant commercial potential for the platform, and this I2I project will firmly establish the platform as a market-ready technology fit for investment and further commercialization. We expect our platform will open new avenues of proteomics research in academia and industry, create new jobs, and grow the Canadian immunoassay industry.