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Rapid homogeneous antibody-based detection of proteins

基于均质抗体的蛋白质快速检测

基本信息

批准号：
7220119
负责人：
TOMASZ HEYDUK
金额：
$ 12.95万
依托单位：
MEDIOMICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2009-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7220119
关键词：
Accident and Emergency department Antibodies Antigens Biological Biological Assay Biological Markers Biomedical Research Cardiac Caring Characteristics Chest Pain Compatible Complex Detection Diagnosis Diagnostic Enzyme-Linked Immunosorbent Assay Epitopes Fluorescence Fluorescence Resonance Energy Transfer Fluorochrome Goals Gold Label Medical Research Methodology Modeling Molecular Nucleic Acids Oligonucleotides Optics Patients Peptide antibodies Peptides Phase Procedures Property Proteins Reading Relative (related person)Reporting Reproducibility Research Sampling Sensitivity and Specificity Series Signal Transduction Specificity Standards of Weights and Measures System Techniques Testing Time Troponin Work antigen antibody binding base concept cost design human disease point of care research study synthetic peptide tool

项目摘要

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.

描述（由申请人提供）：使用利用靶蛋白特异性抗体的各种技术检测特定蛋白质的存在和确定其量是生物医学研究、医学诊断和生物威胁剂检测中最重要的工具之一。这些技术（例如，ELISA）利用了抗体与抗原结合的精确特异性，并构成了目前特异性蛋白质检测的金标准。虽然这些现有技术非常有用，但有一些局限性。一个重要的限制是进行测定所需的时间相对较长。这种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测定的相对技术复杂性，这限制了它们的护理点适用性以及它们对预期最终用户及其环境的需求的适应和定制。该项目的主要目标是开发一种新的基于抗体的蛋白质检测方法（分子钳），该方法将保留经典基于抗体的测定的所有优点，同时减少测定的时间、复杂性和成本。这种新方法是我们以前开发的基于核酸的“分子信标”方法的延伸。这种新的测定形式是均匀的，不需要复杂的样品操作。只需将样品加入到测定混合物中，经过短暂孵育后，易于读取的光学信号（荧光）报告目标分子的存在。虽然分子钳将适用于涉及基于抗体的抗原检测的任何情况，但其独特的性质和特征使其特别适用于需要快速结果的情况。在该项目的第一阶段，我们将使用心肌肌钙蛋白作为模型靶蛋白来测试分子钳的概念，心肌肌钙蛋白是一种心脏生物标志物的优秀例子，需要在胸痛患者的急诊室进行快速测定。虽然I期的所有拟议研究都将涉及心肌肌钙蛋白作为模型，但与分子钳设计相关的经验教训通常适用于其他系统。在第一阶段成功完成后，在第二阶段，将开发、测试和验证针对广泛蛋白质组的分子钳。在第二阶段，将优先考虑目标蛋白质，并确定其快速测定的需求。第II阶段的其他目标是开发与分子钳设计兼容的信号放大程序，以扩展测定的灵敏度。在本提案的第一阶段，我们将测试和验证两种分子钳设计：目标#1：开发和测试基于识别肌钙蛋白两个非结合表位的两种抗体检测肌钙蛋白的分子钳测定。目标二：建立并验证基于单抗体的分子钳法检测肌钙蛋白，该方法基于肌钙蛋白与含表位的合成肽竞争。上述目标的成功实现将验证分子钳设计的实际可行性。我们预计分子钳将作为诊断工具找到非常广泛的应用，允许快速和直接测试人类疾病的生物标志物，并作为研究工具，在可能的情况下，用更简单的进行均相测定代替ELISA测试。