Rapid homogeneous antibody-based detection of proteins

描述(由申请人提供)：利用针对目标蛋白质的抗体的各种技术检测特定蛋白质的存在并确定其数量是生物医学研究、医学诊断和生物威胁物质检测中最重要的工具之一。这些技术(例如，酶联免疫吸附试验)利用了抗体与抗原结合的高度特异性，构成了目前检测特定蛋白的金标准。虽然这些现有技术非常有用，但也有一些局限性。一个重要的限制是进行化验所需的时间相对较长。这一限制在需要立即知道答案的情况下尤其重要，例如，在确定对急诊室护理决策重要的生物标志物时。另一个重要的限制是这些分析的相对技术复杂性，这限制了它们的护理点适用性以及它们的适应和适应预期的最终用户及其环境的需要。该项目的主要目标将是开发一种新的基于抗体的蛋白质检测方法(分子夹子)，它将保留传统基于抗体的分析的所有优点，同时降低分析的时间、复杂性和成本。这种新的方法学是我们以前开发的基于核酸的“分子信标”方法学的延伸。这种新的分析形式是均匀的，不需要复杂的样品处理。只需将样品添加到分析混合物中，在短暂孵育后，一个易于读取的光学信号(荧光)报告目标分子的存在。虽然分子钳子将适用于任何涉及基于抗体的抗原检测的情况，但其独特的性质和特性使其特别适用于需要快速结果的情况。在这个项目的第一阶段，我们将使用心肌肌钙蛋白作为模型目标蛋白，以测试分子钳子的概念。心肌肌钙蛋白是一种在急诊室需要在胸痛患者中快速测定的心脏生物标志物的优秀例子。虽然第一阶段的所有拟议研究都将涉及心肌肌钙蛋白作为模型，但与分子钳子设计相关的经验教训将普遍适用于其他系统。在第一阶段成功完成后，将在第二阶段开发、测试和验证一系列蛋白质的分子钳子。在第二阶段，将优先考虑已确定需要快速测定的目标蛋白质。第二阶段的另一个目标是开发与分子夹设计兼容的信号放大程序，以扩大分析的灵敏度。在这项提案的第一阶段，我们将测试和验证两种分子夹子的设计：目的#1：建立和测试基于识别肌钙蛋白两个非重叠表位的两种抗体的检测肌钙蛋白的分子夹子方法。目的#2：基于肌钙蛋白与含有表位的合成肽的竞争，建立并检测基于单一抗体的分子夹心法。上述目标的成功实现将验证分子钳设计的实际可行性。我们预计，分子夹子将作为一种诊断工具得到非常广泛的应用，允许对人类疾病的生物标记物进行快速和直接的测试，并作为一种研究工具，如果可能的话，用一种更简单的、进行均质分析的方法取代ELISA测试。

DESCRIPTION (provided by applicant): Detection of the presence and the determination of the amount of a specific protein using various techniques utilizing antibodies specific to the target protein are one of the most important tools in biomedical research, medical diagnosis and detection of biological threat agents. These techniques (such as, for example, ELISA) take advantage of the exquisite specificity of antigen binding by the antibodies and constitute the current gold-standard of specific protein detection. While extremely useful, these existing techniques have some limitations. One important limitation is relatively long time required to perform the assay. This limitation is especially important in situations where the answers need to be known immediately such as, for example, in determination of biomarkers important for emergency room care decisions. Another important limitation is a relative technical complexity of these assays which limits their point-of-care applicability and their adaptation and tailoring to the needs of the intended end-users and their settings. The major goal of this project will be to develop a new antibody-based protein detection methodology (molecular pincers), which will retain all advantages of classical antibody-based assays while reducing the time, complexity, and the cost of the assay. This new methodology is an extension of nucleic-acid-based "molecular beacon" methodologies previously developed by us. This new assay format is homogeneous requiring no complicated sample manipulations. A sample needs only to be added to the assay mixture and after a short incubation an easy to read optical signal (fluorescence) reports the presence of the target molecule. While the molecular pincers will be applicable to any situation involving antibody-based detection of antigens, their unique properties and characteristics make them especially applicable in situations requiring rapid results. In Phase I of this project we will use cardiac troponin, an excellent example of a cardiac biomarker requiring quick determination in emergency room in patients with chest pains, as a model target protein to test the concept of molecular pincers. While all of the proposed research in Phase I will involve cardiac troponin as a model, the lessons related to molecular pincer design will be generally applicable to other systems. Following successful completion of Phase I, in Phase II molecular pincers to an extensive panel of proteins will be developed, tested and validated. A priority in Phase II will be given to target proteins with an established need for their rapid determination. Additional goal of Phase II will be to develop signal amplification procedures compatible with molecular pincer design to extend sensitivity of the assay. In Phase I of this proposal we will test and validate two designs of molecular pincers: Aim #1: To develop and test molecular pincer assay for detecting troponin based on two antibodies recognizing two nonoverlaping epitopes of troponin. Aim #2: To develop and test single antibody-based molecular pincer assay for detecting troponin based on competition between the protein and the epitope-containing synthetic peptide. Successful completion of the above aims will validate the practical feasibility of molecular pincer design. We expect that molecular pincers will find very broad applications as a diagnostic tool allowing rapid and straightforward tests for biomarkers of human disease and as a research tool replacing, when possible, ELISA tests with a much simpler to perform homogeneous assay.