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

摘要/摘要 基于血液的多生物标志物面板为有效的癌症早期检测和全人群检测带来了巨大的希望 放映。例如，由三个或更多表现出高灵敏度和特异度的生物标志物组成的板 已经报道了几种不同的癌症，包括卵巢癌、肺癌、乳腺癌和结直肠癌。各种各样的 小型化的固相免疫分析平台也被开发出来，以进行高通量和低成本的检测。 成本多元生物标志物检测与量化。其中包括基于微阵列的仪器， 微流控和微珠技术。 现在的当务之急是将有希望的基于血液的多生物标记物癌症面板过渡到多平台 用于大规模生物标记物的验证和临床最终使用。多重分析技术尤其是 重要的是提供全人口筛查所需的成本效益和高吞吐能力。 然而，多重免疫分析的一个主要问题是所谓的“基质效应”。与大多数人相比 传统的单链测定法，如酶联免疫吸附试验、小型化多重测定法等都非常容易受到干扰。 是由于血液中含有更多的非靶标物质所致。这类干扰可能源自 多种机制包括：i)低特异性异嗜性抗体；ii)基质诱导的珠凝集(例如： 在Luminex®分析中)和iii)非靶基质成分与任何成分的特定或非特异结合 生物标记物分析的结果。此外，样品基质的高粘度(例如，总蛋白浓度高) 会干扰常用于多重检测的微流体。重要的是，基质效应不仅 限制了检测灵敏度，但降低了线性和定量准确性。 在第一阶段，我们将评估一种新的多重血清学癌症检测方法，称为PC-纯™，它是 旨在消除基质效应。这项技术是基于新型可光裂解(PC)连接体的使用 由AmberGen开发，并入亲和力捕获剂，如适配子或抗体。这个 然后将可光解捕捉剂绑在微珠、亲和树脂或其他表面上，并用于分离 目标生物标记物。生物标记物-[捕捉剂]复合体在几分钟内温和而快速地被光释放 在非变性条件下，通过低强度的近紫外光进入定义良好的缓冲区，使同时 多重免疫分析前的目标生物标记物的预纯化和浓缩。与传统不同 使用阻挡缓冲剂、稀释剂和特定基质成分的选择性耗尽的方法，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影响。 在第一阶段，我们将评估PC-Pure™的应用，以改进基于血液的板材的多重检测 肿瘤脱落蛋白生物标记物。肿瘤脱落生物标记物具有高癌症特异性的巨大潜力，但 在血液中发现的丰度极低，因此受到基质效应的影响最大。模型5-生物标记物 将对卵巢癌诊断的蛋白质板进行测试。两个已知浓度的尖峰样本 生物标志物和卵巢癌患者血液样本将在多路Luminex®MAGPIX®平台上进行分析 在第二阶段，在Bio-Plex 2200平台上，设计用于高通量临床测试。 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 对数据进行统计分析的专家指导。我们还将与创始人兼CSO比尔·杰克逊博士密切合作 碱基对生物技术公司的一位研究适配子的领先专家。为了加快PC-纯™的商业化进程，我们将 与多重分析平台的领先制造商Luminex®密切合作(见支持函)。

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).