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)的血清学测试，如PSA和AFP，是简单且经济有效的诊断和预后测试。然而，只有为数不多的有用的血清肿瘤标志物检测可用于临床。最近的临床蛋白质组学表明，即使在癌症的早期发展过程中，血清中也存在特定的生物标记物或蛋白质信号。然而，这些生物标记物在血清中的水平很低，很可能超出了常规ELISA的检测限度。原理研究人员发明了一种新的抗原检测和定量方法，称为T7RNA聚合酶技术催化的荧光放大(FACTT)。FACTT使用与ELISA类似的原理，但结合了T7RNA聚合酶的线性扩增能力。这是一种高通量的等温抗原定量检测方法，初步数据表明，FACTT使ELISA的检测限提高了三个或更多个数量级。FACTT分析是用96孔或384孔板开发的，可以很容易地在临床化学实验室中使用。作为原则的证明，这项应用将专注于开发FACTT检测方法来检测与恶性黑色素瘤相关的循环生物标记物，因为黑色素瘤具有可以靶向的细胞系特异性标记物，但缺乏有用的血清学测试。PIS计划：1)以纯蛋白质或细胞裂解物为模板，建立和优化已知黑色素瘤标志物(酪氨酸酶、Trp-1和Melan-A)的FACTT检测；2)优化血清样本中的FACTT检测，并测试这些检测的稳健性和重复性；然后对从可测量的转移性黑色素瘤患者收集的血清中的肿瘤标志物进行定量。3)验证FACTT分析的特异性，并在大量对照人群中建立分析物的正常范围。在肿瘤进展的早期阶段使用FACTT检测循环生物标志物将使临床医生能够识别高危患者，对患者进行特定治疗的分层，并监测治疗效果和疾病复发。转移性黑色素瘤的早期发现可能会刺激辅助治疗，帮助在临床明显之前根除转移。随着新出现的黑色素瘤治疗选择，如BRAF抑制剂和免疫疗法，迫切需要超灵敏的检测方法来检测早期黑色素瘤的转移。 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: