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记物组合为有效的早期癌症检测和全人群提供了巨大的希望 例如，由三种或更多种具有高灵敏度和特异性的生物标志物组成的组。 据报道，它可治疗多种不同的癌症，包括卵巢癌、肺癌、乳腺癌和结直肠癌。 还开发了小型化固相免疫分析平台来执行高通量和低分析 成本多重生物标志物检测和定量，其中包括基于微阵列的仪器， 微流体和微珠技术。 现在的当务之急是将有前景的基于血液的多生物标志物癌症组合转移到多重平台 对于大规模生物标志物验证和最终在临床中的应用尤其重要。 对于提供全人群筛查所需的成本效益和高通量能力非常重要。 然而，与大多数方法相比，多重免疫分析的一个主要问题是所谓的“基质效应”。 传统的单重检测（例如 ELISA）、小型多重检测非常容易受到干扰 由血液中存在更丰富的非目标物质引起的这种干扰可能源于以下因素。 多种机制，包括：i) 低特异性异嗜性抗体；ii) 基质诱导的珠子聚集（例如 在 Luminex® 检测中）和 iii) 非目标基质成分与任何成分的特异性或非特异性结合 此外，样品基质的高粘度（例如来自高总蛋白浓度）。 可能会干扰多重检测中常用的微流体，重要的是，基质效应不仅会干扰。 限制了测定灵敏度，但降低了线性和定量准确性。 在第一阶段，我们将评估一种称为 PC-PURE 检测™的多重血清学癌症新方法，该方法是 该技术旨在消除基质效应，基于新型光可裂解 (PC) 连接体的使用。 由 AmberGen 开发，可掺入亲和捕获剂（如适体或抗体）中。 然后将可光裂解的捕获剂束缚在微珠、亲和树脂或其他表面上，并用于分离 目标生物标记物-[捕获剂]复合物在几分钟内温和快速地光释放。 在非变性条件下，通过低强度近紫外光进入明确的缓冲区，从而能够同时进行 与传统方法不同，在多重免疫分析之前对目标生物标志物进行预纯化和浓缩。 使用封闭缓冲液、稀释剂和选择性去除的方法，针对特定的基质组分，PC- PURE™ 通过快速预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™ 的应用，以改进基于血液的检测的多重检测 肿瘤脱落蛋白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，因此受基质效应影响最大。模型 5 生物标志物。 将测试用于卵巢癌诊断的蛋白质组。 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 并在专为高通量临床测试而设计的 Bio-Plex 2200 平台上进行第二阶段。 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咨询小组主任，他将提供 我们还将与创始人兼 CSO Bill Jackson 博士密切合作。 为了加速 PC-PURE™ 的商业化，我们将致力于加速 PC-PURE™ 的商业化。 与多重检测平台的领先制造商之一 Luminex® 密切合作（请参阅支持信）。