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Quantitative molecular profiling of tumor biomarkers with multi-color monovalent

多色单价肿瘤生物标志物的定量分子分析

基本信息

批准号：
8307991
负责人：
Hong Yan Liu
金额：
$ 6.2万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8307991
关键词：
3-Dimensional Achievement Acrylamides Affinity Amines Antibodies Antigens Area Binding Binding Sites Biological Biological Markers Biological Models Biology Biopsy Biotin Biotinylation Buffers Cancer Patient Cell Nucleus Cell membrane Cells Cleaved cell Color Cytoplasm DNA Detection Development Devices Diagnosis Disease EZH2 gene Electrostatics Excision Fluorescein-5-isothiocyanate Fluorescence Fluorescent Dyes Formalin Gel Gene Proteins Generations Goals Human Hybrids Image Immunohistochemistry In Vitro Incubated Individual Insulin-Like-Growth Factor I Receptor Ki-67 Antigen Label Left Life Ligands Link Location MME gene Maleimides Malignant Neoplasms Malignant neoplasm of prostate Measurement Methods Mission Molecular Molecular Profiling Nanotechnology Needles PC3 cell line Paraffin Embedding Pathology Patients Peroxidases Play Portraits Primary Neoplasm Procedures Process Protein Microchips Proteins Quantum Dots Reducing Agents Research Research Personnel Resolution Retrieval Role Scientist Secondary to Sepharose Signal Transduction Site Specificity Specimen Staining method Stains Streptavidin Sulfhydryl Compounds System Testing Time Tissues Translating Translations Tumor Markers United States National Institutes of Health Variant anticancer research antigen binding arm base cancer diagnosis cancer therapy cell fixing clinically relevant covalent bond data acquisition design disulfide bond gel electrophoresis in vivo interest meetings migration nanoparticle novel oncology public health relevance rapid detection self assembly spatial relationship survivin tool tumor

项目摘要

DESCRIPTION (provided by applicant): With the development of targeted therapies in cancer, it is imperative to establish methods for analyzing multiple tumor biomarkers accurately and quantitatively in needle tumor biopsies. Accurately and efficiently capturing a specific "molecular portrait'' of each patient's tumor plays a crucial role in the diagnosis and treatment cancers and are challenging the current method of peroxidase-based immunohistochemistry which is not stoichiometric and only one stain can be done on a section. Nanotechnology is becoming a fundamental driver of advances in cancer research. In this regard, bioconjugated quantum dots (QDs) are particularly attractive as an emerging new probe for tumor biomarker profiling owing to its high multiplexing and quantification capabilities. QDs possess unique advantages compared to organic dyes and fluorescent proteins, such as high brightness and photostability, simultaneous excitation of multiple fluorescence colors with a single excitation wavelength. Although QDs have become a new biological labeling entity, the "perfect '' QD bioconjugates, in which the number of biomolecules per nanoparticle and the orientation of the biomolecules are precisely controlled, are still not available and pressing in need for accurate and quantitative detection biomarker expression. The lack of orientation control can cause the loss of QD-ligand activity while the lack of stoichiometric control will bring about variation in signal quantification. The goal of the proposed research is to develop a panel of multi-color orientation controlled monovalent QD probes using hybrid gel electrophoresis. This gel system is highly capable of separating QDs because QDs cannot enter or be fractionated by poly-acrylamide gel. Although QDs can enter agarose gel due to its larger pore size than hybrid gel, QD separation in agarose gel cannot reach the same resolution as hybrid gel can. An antibody contains two antigen binding sites, monovalent antibody containing one antigen binding site is generated by partial reduction of a whole antibody using a reducing agent. Conjugation orientation control is realized through site-specific biotinylating sulfhydryls in the antibody hinge region and leave the antigen binding site intact. Conjugates with one copy of streptavidin per QD and monovalent QD will be separated through the hybrid gel and recovered using a lab-made eluter device. The standard immunohistochemistry procedures will be followed using formalin-fixed and paraffin-embedded (FFPE) prostate cancer cell lines. Hyperpectral imaging system will be applied to perform data acquisition and quantitative analysis. Novel monovalent QD probes will provide an unprecedented precision tool in determining the molecular fingerprints of individual cancers, on which accurate diagnosis and treatment decision can be made. Our results will be relevant to the mission of the NIH and be broadly interesting to researchers studying pathology of a variety of diseases. PUBLIC HEALTH RELEVANCE: This project will conduct tumor biomarker profiling by using ongoing synthesis of a panel of multi-color monovalent Quantum Dot probes, the predicting results of accurate quantitative measurement tumor markers will provide the essential information for cancer diagnosis and personalized treatment. Our research will be relevant to the mission of the NIH and be broadly interesting to researchers studying the pathology of variety diseases.

描述（由申请人提供）：随着癌症靶向治疗的发展，迫切需要建立在针吸肿瘤活检中准确定量分析多种肿瘤生物标志物的方法。准确有效地捕获每个患者肿瘤的特定“分子肖像”在诊断和治疗癌症中起着至关重要的作用，并且正在挑战目前基于过氧化物酶的免疫组织化学方法，该方法不是化学计量的，并且只能在切片上进行一种染色。纳米技术正在成为癌症研究进步的基本驱动力。在这方面，生物共轭量子点（QD）是特别有吸引力的，作为一个新兴的肿瘤生物标志物分析的新探针，由于其高度的多重和定量能力。与有机染料和荧光蛋白相比，量子点具有独特的优势，如高亮度和光稳定性，用单一激发波长同时激发多种荧光颜色。尽管QD已经成为一种新的生物标记实体，但其中每个纳米颗粒的生物分子的数量和生物分子的取向被精确控制的“完美"QD生物缀合物仍然不可用，并且迫切需要精确和定量检测生物标志物表达。取向控制的缺乏会导致QD-配体活性的损失，而化学计量控制的缺乏会导致信号定量的变化。本研究的目的是利用混合凝胶电泳技术开发一种多色取向控制的单价量子点探针。该凝胶系统高度能够分离QD，因为QD不能进入聚丙烯酰胺凝胶或被聚丙烯酰胺凝胶分级。虽然量子点可以进入琼脂糖凝胶，由于其比杂化凝胶更大的孔径，在琼脂糖凝胶中的量子点分离不能达到与杂化凝胶相同的分辨率。抗体含有两个抗原结合位点，含有一个抗原结合位点的单价抗体是通过使用还原剂部分还原整个抗体而产生的。缀合方向控制通过抗体铰链区中的位点特异性生物素化巯基来实现，并使抗原结合位点保持完整。每个QD具有一个链霉亲和素拷贝和单价QD的缀合物将通过混合凝胶分离，并使用实验室制备的洗脱装置回收。将使用福尔马林固定和石蜡包埋（FFPE）前列腺癌细胞系遵循标准免疫组织化学程序。超光谱成像系统将用于进行数据采集和定量分析。新型单价量子点探针将为确定个体癌症的分子指纹提供前所未有的精确工具，从而可以做出准确的诊断和治疗决策。我们的研究结果将与NIH的使命相关，并对研究各种疾病病理学的研究人员产生广泛的兴趣。公共卫生相关性：本项目将利用正在合成的一组多色单价量子点探针进行肿瘤生物标志物谱分析，准确定量测量肿瘤标志物的预测结果将为癌症诊断和个性化治疗提供必要的信息。我们的研究将与美国国立卫生研究院的使命相关，并对研究各种疾病病理学的研究人员产生广泛的兴趣。