Intramolecular Immunoassay for Probing Paracrine Signaling

用于探测旁分泌信号传导的分子内免疫分析

基本信息

批准号：
8001695
负责人：
Robert G Lowery
金额：
$ 25.09万
依托单位：
BELLBROOK LABS, LLC
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 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8001695
关键词：
Amino Acids Antibodies Antigen-Antibody Complex Antineoplastic Agents Architecture Benchmarking Binding Biological Assay Biological Models Cell model Cell physiology Cells Cellular Assay Clinical Trials Complex Cysteine Detection Diffuse Diffusion Elements Engineering Environment Epithelial Cells Epitopes Event Extracellular Matrix Fab Immunoglobulins Fibrinogen Figs - dietary Fluorescence Fluorescence Resonance Energy Transfer Image Immunoassay Immunoglobulin G Individual Label Length Methods Microscopic Modeling Modification Monoclonal Antibodies Non-Invasive Cancer Detection Paracrine Communication Peptides Phase Protein Isoforms Reader Reagent Reporter Resolution Role Screening procedure Series Signal Transduction Signaling Molecule Site Solid Structure Testing Time Tissues Tracer Tumor Biology Vascular Endothelial Growth Factors antigen binding base cell type drug discovery enzyme substrate flexibility human VEGF protein in vitro Assay instrument intercellular communication meetings monolayer novel polypeptide prevent prototype public health relevance success tool tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Key elements of tumorigenesis are driven by paracrine signaling between stromal and epithelial cells within the context of the extracellular matrix. Unfortunately, the cellular assays used for drug discovery typically rely on a single cell type grown as a monolayer, and this aspect of tumor biology very poorly. This may be one factor contributing to the low success rate of anti-cancer drugs in clinical trials. Though cellular models incorporating multiple cell types in a tissue-like environment are available, they are difficult to integrate with current automated microscopic imaging platforms used by pharma for high content analysis of cell function. An especially challenging problem is detection of soluble paracrine signaling factors in extracellular matrix. Though antibodies are available for many of these factors, existing immunoassay methods are not suited for use in a dense matrix. A fundamental limitation is the requirement for multiple components: the primary antibody and secondary reporter reagents. To overcome this limitation we are proposing to develop an intramolecular immunoassay; i.e., an antibody with a built-in fluorescent reporter. We will achieve this by tethering a fluorescent tracer on a flexible polypeptide linker to IgG and Fab fragments engineered with seleno cysteine residues for site specific modification. A well characterized monoclonal antibody for vascular endothelial growth factor (VEGF) will be used as an initial model. A series of prototype intramolecular immunoassay reagents will be assembled and tested for fluorescence based detection of VEGF to arrive at the optimal structure. In Phase II, the intramolecular immunoassay will be combined with BellBrook's novel microconduit array platform, iuvo, to enable dynamic imaging of paracrine signaling at the level of soluble factors secreted from individual cells. PUBLIC HEALTH RELEVANCE: Important aspects of tumorigenesis are controlled by signaling between different cell types that reside in a dense matrix that provides structural support to tissues and also participates in signaling. Unfortunately, it is very difficult to incorporate these signaling events into in vitro assays that can be used to test potential anti-cancer drugs. To overcome this limitation, we propose to develop a novel immunoassay method that would allow probing of cell-cell signaling in a tissue-like matrix using existing automated detection instruments.

描述（由申请人提供）：肿瘤发生的关键要素是由细胞外基质内的基质细胞和上皮细胞之间的旁分泌信号传导驱动的。不幸的是，用于药物发现的细胞测定通常依赖于单层生长的单一细胞类型，而肿瘤生物学的这方面效果非常差。这可能是抗癌药物临床试验成功率低的因素之一。尽管可以在类组织环境中结合多种细胞类型的细胞模型，但它们很难与制药公司当前用于细胞功能高内涵分析的自动显微成像平台集成。一个特别具有挑战性的问题是检测细胞外基质中的可溶性旁分泌信号因子。尽管抗体可用于许多这些因素，但现有的免疫测定方法不适合在致密基质中使用。一个根本的限制是需要多种成分：一抗和二抗报告试剂。为了克服这一限制，我们建议开发一种分子内免疫测定法；即具有内置荧光报告基因的抗体。我们将通过将荧光示踪剂拴在柔性多肽接头上来实现这一目标，该接头与用硒代半胱氨酸残基工程化的 IgG 和 Fab 片段进行位点特异性修饰。一种经过充分表征的血管内皮生长因子（VEGF）单克隆抗体将被用作初始模型。将组装和测试一系列原型分子内免疫测定试剂，用于基于荧光的 VEGF 检测，以获得最佳结构。在第二阶段，分子内免疫测定将与 BellBrook 的新型微导管阵列平台 iuvo 相结合，从而能够在单个细胞分泌的可溶性因子水平上对旁分泌信号进行动态成像。公共卫生相关性：肿瘤发生的重要方面是由不同细胞类型之间的信号传导控制的，这些细胞类型位于致密基质中，为组织提供结构支持并参与信号传导。不幸的是，很难将这些信号事件纳入可用于测试潜在抗癌药物的体外测定中。为了克服这一限制，我们建议开发一种新型免疫测定方法，该方法允许使用现有的自动检测仪器探测组织样基质中的细胞间信号传导。