Photocleavage Technology for Improved Serum-based Multi-Biomarker Cancer Assays

用于改进基于血清的多生物标志物癌症检测的光裂解技术

• 批准号: 9175644
• 负责人: Mark Lim
• 金额: $ 22.5万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175644
• 关键词: Achievement; Affinity; Antibodies; Base Pairing; Binding; Biological Assay; Biological Markers; Biometry; Biotechnology; Blood; Blood specimen; Boston; Buffers; Cancer Patient; Clinic; Collaborations; Colorectal Cancer; Complex; Consult; Data; Detection; Diagnostic Sensitivity; Enzyme-Linked Immunosorbent Assay; Exhibits; Heterophile Antibodies; Immunoassay; Industry Standard; Letters; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Manufacturer Name; Mediating; Microfluidics; Modeling; Phase; Plant Resins; Plasma; Population; Proteins; Public Health Schools; Publishing; Reporting; Reproducibility; Research; Sampling; Screening for Ovarian Cancer; Screening for cancer; Sensitivity and Specificity; Serological; Serum; Solid; Specificity; Staging; Statistical Data Interpretation; Surface; Technology; Testing; Ultraviolet Rays; Universities; Validation; Viscosity; Work; aptamer; base; biomarker panel; cancer biomarkers; cancer diagnosis; commercialization; cost; cost effectiveness; design; falls; improved; instrument; interest; malignant breast neoplasm; mid-career faculty; miniaturize; multiplex detection; novel; novel strategies; protein biomarkers; research clinical testing; research study; screening; targeted biomarker; tumor

SUMMARY/ABSTRACT Blood-based multi-biomarker panels hold great promise for effective early cancer detection and population-wide screening. For example, panels consisting of three or more biomarkers which exhibit high sensitivity and specificity have been reported for several different cancers including ovarian, lung, breast and colorectal cancer. A variety of miniaturized solid-phase immunoassay platforms have also been developed to perform high throughput and low- cost multiplex biomarker detection and quantification. These include instruments based on microarrays, microfluidics and micro-bead technology. A high priority is now to transition promising blood-based multi-biomarker cancer panels to multiplex platforms for large-scale biomarker validation and ultimate use in the clinic. Multiplex assay technologies are especially important to provide the cost-effectiveness and high throughput capacity necessary for population-wide screening. However, a major problem with multiplex immunoassays is the so-called “matrix effect”. Compared to most conventional single-plex assays such as ELISA, miniaturized multiplex assays are highly susceptible to interference caused by the presence of the more abundant, non-target agents in blood. Such interference can originate from a variety of mechanisms including: i) low specificity heterophile antibodies; ii) matrix-induced bead aggregation (e.g. in Luminex® assays) and iii) specific or non-specific binding of non-target matrix components to any component of the biomarker assay. In addition, high viscosity of the sample matrix (e.g. from high total protein concentration) can interfere with the microfluidics commonly used for multiplex assays. Importantly, the matrix effect not only limits assay sensitivity, but reduces linearity and quantitative accuracy. During Phase I we will evaluate a new approach to multiplex serological cancer assays termed PC-PURE™ which is designed to eliminate the matrix effect. This technology is based on the use of novel photocleavable (PC) linkers developed by AmberGen which are incorporated into affinity capture agents such as aptamers or antibodies. The photocleavable capture agents are then tethered to micro-beads, affinity resins or other surfaces and used to isolate the target biomarker. The biomarker-[capture agent] complexes are gently and rapidly photo-released in minutes under non-denaturing conditions by low-intensity near-UV light into a well-defined buffer, enabling simultaneous pre-purification and concentration of the target biomarkers prior to multiplex immunoassay. Unlike conventional approaches using blocking buffers, diluents and selected depletion, specific to particular matrix components, PC- PURE™ eliminates all matrix effects by rapidly pre-purifying the biomarkers of interest. In Phase I we will evaluate the application of PC-PURE™ to improve the multiplex detection of blood-based panels of tumor-shed protein biomarkers. Tumor-shed biomarkers have great potential for high cancer specificity but are found at extremely low abundance in the blood and hence suffer most from the matrix effect. A model 5-biomarker protein panel for ovarian cancer diagnosis will be tested. Both spike-in samples with known concentrations of the biomarkers and ovarian cancer patient blood samples will be analyzed on a multiplex Luminex® MagPix® platform and in Phase II on a Bio-Plex 2200 platform designed for high-throughput clinical testing. This research will be conducted in collaboration with Dr. Gheorghe Doros, Associate Professor of Biostatistics and Director of the Biostatistics Consulting Group at the Boston University School of Public Health, who will provide expert guidance for statistical analysis of the data. We will also work closely with Dr. Bill Jackson, Founder and CSO of Base Pair Biotechnologies, a leading expert on aptamers. To accelerate commercialization of PC-PURE™, we will work closely with Luminex®, one of the leading manufacturers of multiplex assay platforms (see letters of support).

摘要/摘要 基于血液的多生物标志物面板对有效的早期癌症检测和范围内有很大的希望 筛选。例如，由三个或多个生物标志物组成的面板暴露了高灵敏度和特异性 据报道，包括卵巢，肺，乳腺癌和大肠癌在内的几种不同的癌症。各种各样 还开发了微型固相免疫测定平台，以执行高吞吐量和低 - 成本多重生物标志物检测和定量。这些包括基于微阵列的仪器， 微流体和微珠技术。 现在的高度优先级是过渡有希望的血液基多生物标志物癌面板到多重平台 用于诊所中的大规模生物标志物验证和最终使用。多重测定技术尤其是 重要的是提供整个人口筛查所需的成本效益和高吞吐能力。 但是，多重免疫测定的一个主要问题是所谓的“矩阵效应”。与大多数 常规的单重测定，例如ELISA，微型多重分析非常容易受到干扰 是由于血液中更丰富，非目标剂的存在引起的。这种干扰可以起源于 多种机制，包括：i）低特异性杂抗抗体； ii）基质诱导的珠聚集（例如 在Luminex®分析中）和iii）非目标基质组件与任何组件的特定或非特异性结合 生物标志物测定法。此外，样品基质的高粘度（例如，来自高蛋白质浓度） 可以干扰通常用于多重测定的微流体。重要的是，矩阵效应不仅 限制敏感性，但降低了线性和定量准确性。 在第一阶段，我们将评估一种新方法，用于称为PC-PURE™的多重血清癌测定法 旨在消除矩阵效果。该技术基于新颖的光电启动（PC）链接器的使用 由Ambergen开发的，该Ambergen被纳入亲和捕获剂，例如适体或抗体。这 然后，将光电捕获剂束缚在微珠，亲和力树脂或其他表面上，并用于隔离 目标生物标志物。生物标志物 - [捕获剂]复合物在几分钟内轻轻而快速拍摄 在不掩盖的条件下，低强度近紫外光进入一个定义明确的缓冲液，可以同时进行 在多重免疫测定之前，目标生物标志物的预纯化和浓度。与传统不同 使用阻止缓冲液，稀释剂和选定部署的方法，特定于特定矩阵组件，PC- Pure™通过快速预先纯化感兴趣的生物标志物来消除所有基质效应。 在第一阶段，我们将评估PC-Pure™在改善血液基面板的多重检测中的应用 肿瘤蛋白生物标志物的。肿瘤分裂生物标志物具有高癌症特异性的巨大潜力，但 血液中的丰度极低，因此遭受基质效应的最大影响。 5个模型标记物 将测试用于卵巢癌诊断的蛋白质面板。两种峰值样品都有已知浓度的 生物标志物和卵巢癌患者血液样本将在多重Luminex®Magpix®平台上进行分析 在II阶段，在Bio-Plex 2200平台上，专为高通量临床测试而设计。 这项研究将与生物统计学副教授Gheorghe Doros博士合作进行 波士顿大学公共卫生学院生物统计学咨询小组主任，他将提供 数据统计分析的专家指南。我们还将与创始人兼CSO Bill Jackson博士紧密合作 基本对生物技术，适体领域的主要专家。为了加速PC-Pure™的商业化，我们将 与多路复用测定平台的领先制造商之一Luminex®紧密合作（请参阅支持信）。