Rapid homogeneous antibody-based detection of proteins

描述（由申请人提供）：检测和定量各种生物样品中的蛋白质是最重要和最常用的测定法之一，其应用于研究、医学诊断、早期疾病检测和生物威胁因子检测。虽然有许多蛋白质检测方法可用（ELISA是其中的金标准），但它们各自具有一些局限性。最常见的限制是进行测定所需的时间和测定中涉及的样品操作的复杂性。因此，显然需要开发新的快速和同质的蛋白质检测方法。在该项目的第一阶段，我们建立了一种新的基于抗体的同质蛋白检测方法（分子钳）的可行性，该方法允许通过简单的荧光强度测量来检测靶蛋白的存在。该检测方法快速，技术上非常简单（除了将样品与检测试剂混合外，基本上不涉及样品操作）。这些特征使得这种新的测定形式在其中快速测定靶蛋白是必需的而没有配备有复杂仪器的实验室的支持的情况下（例如，在护理点诊断测定的情况下）特别有用。该项目的长期目标是利用分子钳为一组心脏病标志物开发简单，快速和廉价的即时诊断测试。该II期项目的目标是为研究市场开发6种心脏病标志物（肌钙蛋白I、B型利钠肽（BNP）、肌红蛋白、肌酸激酶（CK-MB）、C反应蛋白（CRP）和D-二聚体）的分子钳测定法。这一目标的完成将允许启动批准这些检测试剂盒用于临床诊断的过程。此外，我们将致力于通过开发策略来提高分析灵敏度，从而增强分子钳分析。这一目标的完成将通过扩大可开发测定的可能靶点的范围来增强分子钳的商业化前景。 公共卫生相关性：分子钳测定将在人类疾病的研究和诊断中得到广泛应用。在研究中，它可以成为更方便，更容易使用，更便宜的替代目前使用的检测蛋白质的方法。在诊断中，它将允许更快速和更直接地检测与人类疾病相关的抗原。

DESCRIPTION (provided by applicant): Detection and quantification of proteins in various biological samples is one of the most important and most often utilized assays with applications in research, medical diagnosis, early disease detection and detection of biological threat agents. While many protein detection methodologies are available (ELISA being a gold standard of those), they each have some limitations. The most common limitation is the time required to perform the assay and the complexity of the sample manipulation involved in the assay. Thus, there is a clear need for developing new rapid and homogenous protein detection methodologies. In Phase I of this project we established feasibility of a novel antibody-based homogenous protein detection methodology (molecular pincers) which allows detection of the presence of a target protein by a simple fluorescence intensity measurement. The assay is rapid and is technically extremely simple to perform (involves essentially no sample manipulation other then mixing the sample with the detection reagent). These characteristics make this new assay format particularly useful in situations where quick determination of the target protein is essential without a support of a laboratory equipped with sophisticated instrumentation (as in the case, for example, of point-of-care diagnostic assay). The long-term goal of this project will be to utilize molecular pincers to develop simple, rapid and inexpensive point-of-care diagnostic tests for a panel of cardiac disease markers. The goal of this Phase II project will be to develop molecular pincer assays for six markers of cardiac disease (troponin I, B-type natriuretic peptide (BNP), myoglobin, creatine kinase (CK-MB), C-reactive protein (CRP), and D-dimer) for research market. Completion of this goal will allow initiation of the process of approval of these assays for clinical diagnosis. Additionally, we will work on enhancing molecular pincer assay by developing strategies to improve assay sensitivity. Completion of this goal will enhance the commercialization prospects of molecular pincers by broadening the range of possible targets for which the assay could be developed. PUBLIC HEALTH RELEVANCE: Molecular pincer assays will find wide applications in research and diagnosis of human disease. In research, it could become more convenient, easier to use, less expensive replacement for currently used assays for detecting proteins. In diagnosis, it will allow more rapid and more straightforward detection of antigens correlated with human disease.