Multiplexed Ultrasensitive Mix-and-Read Tumor Marker Quantification Assays

描述（由申请人提供）：Nesher Technologies，Inc. (NTI)从加州大学洛杉矶分校授权的知识产权的革命性套件的超灵敏度生物检测技术，涉及交替激光激发（ALEX）荧光光谱。这些技术是在加州大学洛杉矶分校单分子生物物理实验室（由Shimon韦斯教授领导）开发的。NTI建议将这些技术之一应用于非常早期的癌症诊断，监测治疗反应，评估结果，预测复发以及基础癌症研究。在被称为交替激光激发荧光辅助分子分选（ALEX-FAMS）的拟议实施方案中，两种识别分子用不同颜色的荧光染料标记。在飞升共焦检测体积中的两种颜色的重合检测构成阳性目标检测事件。该提案的广泛的长期目标是开发高度多路复用、混合和读取、高通量溶液相测定，用于快速和超灵敏（飞摩尔）定量血清、尿液和唾液中肿瘤标志物的蛋白质和mRNA水平（每个标准患者样品具有> 10，000个标志物的能力），克服当前技术的缺点并使成本最小化。在资助期间，我们将开发一种基于蛋白质的检测方法，使用两种高亲和力荧光标记抗体检测癌胚抗原（CEA），并证明我们方法的定量性质。我们的具体目标是： 1. CEA 2高亲和力抗体的表达和纯化 2.两种不同荧光染料与这些抗体的缀合 3. ALEX单分子荧光光谱法检测癌胚抗原的评价 开发超灵敏、低成本的诊断检测方法，通过允许从单个患者样本中同时检测和准确定量数千种肿瘤标志物，从根本上突破了当前技术的极限，将有助于临床医生和癌症研究人员提高肿瘤标志物分析的有效性。定期监测公众与癌症有关的体液变化的前景将大大降低死亡率，并有助于对抗这种致命的疾病。

DESCRIPTION (provided by applicant): Nesher Technologies, Inc. (NTI) licensed from UCLA the IP for a revolutionary suite of ultrasensitive biodetection technologies involving alternating laser excitation (ALEX) fluorescence spectroscopy. These technologies were developed at the UCLA Single Molecule Biophysics Laboratory (headed by Prof. Shimon Weiss). NTI proposes to apply one of these technologies for very early-stage cancer diagnosis, monitoring of therapeutic response, assessment of outcome, prediction of recurrence, as well as basic cancer research. In the proposed implementation, dubbed alternating-laser excitation fluorescence-aided molecule sorting, or ALEX-FAMS, two recognition molecules are tagged with different color fluorescence dyes. Coincident detection of the two colors in a femto-liter confocal detection volume constitutes a positive target detection event. The proposal's broad long-term objective is development of highly multiplexed, mix-and-read, high- throughput solution phase assays for rapid and ultra-sensitive (femtomolar) quantification of protein and mRNA levels of tumor markers in serum, urine, and saliva (with a capacity of >10,000 markers per standard patient sample), overcoming drawbacks of current technologies and minimizing costs. Over the funding period, we will develop a protein-based assay using two high affinity fluorescently labeled antibodies to detect carcino-embryonic antigen (CEA) and demonstrate the quantitative nature of our methodology. Our specific aims are: 1. Expression and purification of a high-affinity antibody pair specific for CEA 2. 2. Conjugation of two different fluorescent dyes to these antibodies 3. Evaluation of CEA detection with ALEX-based single molecule fluorescence spectroscopy Development of ultra-sensitive, low-cost diagnostic assays that radically push the limits of current technologies by allowing simultaneous detection and accurate quantification of thousands of tumor markers from a single patient sample will aid both clinicians and cancer researchers in improving usefulness of tumor marker analysis. The prospect of monitoring the public on a routine basis for cancer-related changes in bodily fluids will significantly decrease mortality rates and and help fighting this deadly disease.