Ultrasensitive FACTT assays for serum biomarkers

描述（由申请人提供）：循环肿瘤标志物（生物标志物）（如PSA和AFP）的血清学检测是简单且具有成本效益的诊断和预后检测。然而，只有少数有用的血清肿瘤标志物测试可用于临床。最近的临床蛋白质组学表明，即使在早期癌症发展期间，血清中也存在特定的生物标志物或蛋白质特征。然而，这些生物标志物以低水平存在于血清中，并且可能超过常规ELISA的检测限。主要研究人员发明了一种新的抗原检测和定量方法，称为T7 RNA聚合酶催化荧光扩增技术（FACTT）。FACTT使用与ELISA类似的原理，但结合了T7 RNA聚合酶的线性扩增能力。这是一种高通量等温定量抗原检测方法，初步数据显示FACTT使ELISA的检测限提高了三个或更多个数量级。FACTT测定使用96孔或384孔板开发，并且可以容易地用于临床化学实验室。作为原理证明，本申请将集中于开发FACTT测定以检测与恶性黑素瘤相关的循环生物标志物，因为黑素瘤具有可以靶向的细胞谱系特异性标志物，但缺乏有用的血清学测试。Pis计划：1）使用纯蛋白质或细胞裂解物作为模板，建立并优化已知黑素瘤标志物（酪氨酸酶、TRP-1和Melan-A）的FACTT测定; 2）优化血清样品中的FACTT测定，并测试这些测定的稳健性和再现性;然后定量从患有可测量的转移性黑素瘤的患者收集的血清中的肿瘤标志物。3)验证FACTT检测试剂盒的特异性，并在大型对照人群队列中建立分析物的正常范围。使用FACTT测定在肿瘤进展的早期阶段检测循环生物标志物将允许临床医生识别高风险患者，对患者进行分层以进行特定治疗，并监测治疗疗效和疾病复发。转移性黑色素瘤的早期检测可能会刺激辅助治疗，有助于在转移变得临床明显之前根除转移。随着Braf抑制剂和免疫疗法等黑色素瘤治疗方案的出现，迫切需要超灵敏的检测方法来检测早期黑色素瘤转移。 评估：

DESCRIPTION (provided by applicant): Serological tests for circulating tumor markers (biomarkers), such as PSA and AFP, are simple and cost-effective diagnostic and prognostic tests. However, only a handful of useful serum tumor marker tests are available clinically. Recent clinical proteomics indicates that specific biomarkers or protein signatures are present in the serum even during early cancer development. However, these biomarkers are present in the serum in low levels and likely beyond the detection limit of conventional ELISA. The principle investigators have invented a new antigen detection and quantification method termed Fluorescent Amplification Catalyzed by T7 RNA polymerase Technology (FACTT). FACTT uses similar principles as ELISA but coupled with the linear amplification ability of T7 RNA polymerase. It is a high throughput isothermal quantitative antigen detection assay and the preliminary data showed that FACTT increased the detection limit of ELISA by three or more orders of magnitude. FACTT assays are developed using 96 well or 384 well plates and can be easily used in clinical chemistry laboratory. As a proof of principle, this application will focus on developing FACTT assays to detect circulating biomarkers associated with malignant melanomas, because melanomas have cell lineage specific markers that can be targeted, but useful serological tests are lacking. The Pis plan to: 1) Set up and optimize FACTT assays to known melanoma markers (tyrosinase, TRP-1 and Melan-A) using pure proteins or cell lysates as template; 2) Optimize the FACTT assays in serum samples and test robustness and reproducibility of these assays; and then quantify tumor markers in sera collected from patients with measurable metastatic melanoma. 3) Validate specificity of the FACTT assays and establish normal ranges of the analytes in large cohorts of control population. The detection of circulating biomarkers in the early stage of tumor progression using FACTT assays would allow clinicians to identify high risk patients, to stratify patients for specific treatment, and to monitor treatment efficacy and disease recurrence. Early detection of metastatic melanoma might spur adjuvant therapies that could help eradicate metastases before they become clinical evident. With the emerging treatment options for melanoma such as Braf inhibitors and immunotherapies, there is a strong need for ultrasensitive assays to detect early melanoma metastasis. ASSESSMENT: