Utrasensitive bioassays using palladium catalyzed chemical amplification

描述（由申请人提供）：我们提议使用新型钯催化化学扩增（PdCCA）技术开发超灵敏生物测定法，该方法可检测血液中极低浓度的肿瘤生物标志物。PdCCA是一种通用技术，可普遍应用于不同的分析平台。在这里考虑的生物测定应用中，其使用用钯催化剂标记的检测抗体和随后的钯催化的高增益氧化还原反应，产生水溶性甲瓒染料。PdCCA将适用于标准ELISA形式，其中它可以在免疫测定中提供102至103倍的灵敏度增加因子，其中可获得高选择性的检测抗体。这种灵敏度的增加将降低目前使用的方法的检测阈值至ng-pg/ml的期望范围，从而允许开发用于早期肿瘤检测的筛选生物标志物。拟议工作的第一阶段将集中于技术开发和优化，使用一组卵巢癌的三种生物标志物和任选的乳腺癌生物标志物，目前正在华盛顿州西雅图的弗雷德哈钦森癌症研究中心（FHCRC）进行研究。我们将优化染料形成扩增化学和分光光度检测，并使用体外试验模型来开发和优化生物测定技术。我们的方法的可行性将在FHCRC储存的临床标本上得到证明。 公共卫生相关性：在早期无症状阶段检测癌症可以显著提高患者的生存率，但目前还没有简单可靠的分子诊断癌症筛查测试。即使在早期阶段，肿瘤也会分泌特定的分子生物标志物，但它们在体液中的浓度通常显著低于目前可用的检测方法的灵敏度。更灵敏的检测平台将有助于开发用于早期癌症诊断和治疗的简单、非侵入性筛查测试，从而提高患者的生存率。一旦PdCCA的效用被证明为最初的一组生物标志物，各种候选分子肿瘤标志物的新测试将被开发，临床验证和商业化。商业产品将包括生物标志物特异性试剂盒和优化的检测程序。由于我们正在利用完善的ELISA平台，该技术不需要任何新设备，并且对于广泛的临床应用具有商业吸引力。这种检测的可用性将对疾病的早期发现和患者存活率以及控制医疗保健费用产生重大影响。

DESCRIPTION (provided by applicant): We propose to use novel Palladium Catalyzed Chemical Amplification (PdCCA) technology to develop ultra-sensitive bioassays that can detect very low concentrations of tumor biomarkers in blood. PdCCA is a generic technology that can be universally applied to different assay platforms. In the bioassay application considered here it uses a detection antibody labeled with a palladium catalyst and a subsequent, palladium catalyzed, high gain redox reaction generating a water-soluble formazan dye. PdCCA will be adapted to the standard ELISA format, where it may provide a factor of 102 to 103 fold increase in sensitivity in immunoassays for which a detection antibody of high selectivity is available. This increase in sensitivity will lower the detection threshold of currently used methods to the desired range of ng-pg/ml, allowing for development of screening biomarkers for early tumor detection. Phase I of the proposed work will concentrate on technology development and optimization using a set of three biomarkers for ovarian cancer and, optionally, one biomarker of breast cancer, currently under study at the Fred Hutchinson Cancer Research Center, Seattle, WA (FHCRC). We will optimize the dye forming amplification chemistry and spectrophotometric detection, and use model in vitro assays to develop and optimize the bioassay technology. Feasibility of our approach will be demonstrated on clinical specimens stored at the FHCRC. PUBLIC HEALTH RELEVANCE: Detection of cancer in the early, asymptomatic stages dramatically improves patients' survival, but currently there are no simple and reliable molecular diagnostic cancer screening tests. Even at early stages, tumors secrete specific molecular biomarkers, but their concentration in body fluids is typically significantly below the sensitivity of currently available assays. More sensitive detection platforms will facilitate the development of simple, non-invasive screening tests for early cancer diagnosis and treatment, leading to improved patient survival. Once PdCCA utility is demonstrated for the initial set of biomarkers, new tests for a variety of candidate molecular tumor markers will be developed, clinically verified and commercialized. The commercial product will consist of biomarker specific reagent kits and optimized detection procedures. Since we are taking advantage of the well established ELISA platform, this technology does not require any new equipment and is commercially attractive for a broad range of clinical applications. Availability of such tests will have a significant impact on early detection of disease and patient survival rates, as well as containment of the health care costs.