Multiplexed single cell cytokine secretion assay

多重单细胞细胞因子分泌测定

• 批准号: 6833282
• 负责人: WILLIAM D HARRIMAN
• 金额: $ 13.65万
• 依托单位: TRELLIS BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-15 至 2004-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833282
• 关键词: cytokine; enzyme linked immunosorbent assay; helper T lymphocyte; immunologic assay /test; immunoregulation; method development; phenotype

DESCRIPTION (provided by applicant): Analysis of the cytokine network in normal immunoregulation and in disease processes has benefited from the development of the immunospot assay which can interrogate individual cells for secretion of specific immunoreactive antigens. In the ELISPOT version of this assay, cells are settled onto a solid phase surface of capture antibodies for several hours, and the resulting secreted-and-captured antigen is detected by the precipitated product of an enzyme conjugated to a detector antibody. The result is a colored spot at the site of the antigen-secreting cell. This assay has proven to be exquisitely sensitive, as it captures secreted protein immediately after release and prior to diffusion into a larger volume. The assay has also proven to be uniquely informative as it provides valuable information on cell to cell variation that is lost in ELISA assays run on secreted protein from pools of cells. The ELISPOT assay is currently used to characterize several normal immune functions and immune cell diseases, including leukemias, autoimmune conditions, and HIV/AIDS. A major limitation of the ELISPOT assay is that it only looks at one or, at most two, secreted products per cell. Trellis has previously developed a proprietary cell-based multiplex microscopy technology and has now extended it to the ELISPOT resulting in a true profiling immunospot assay, the CelISPOT. The Specific Aims of this Phase I proposal are to: 1. automate the present manual system for reading and classifying CelISPOT cytokine secretion patterns; 2. extend the assay from the current 5-plex murine system to encompass 9 human cytokines; and 3. use the system to classify human T cells according to the conventional Th1 and Th2 phenotypes, and determine the extent to which individual cells deviate from these classes. The overall result of this Phase I effort will be a fully functional platform for a multiplex immuno spot analysis. In Phase II of this project, we will extend the reagent set to 25 cytokines, and together with our external collaborators, we will compile a database of results in major disease indications to support commercialization in Phase IIl. The availability of multiplex immunospot analysis for a comprehensive complement of cytokines, with sufficient sensitivity to detect rare cells, is expected to have far-reaching application in clinical medicine. The CelISPOT assay's unique ability to characterize pathological phenotypes in detail will provide unprecedented precision and sensitivity in characterization of immune system function for a wide range of important diseases.

描述（由申请人提供）：正常免疫调节和疾病过程中细胞因子网络的分析受益于免疫斑点试验的开发，该试验可以询问单个细胞分泌特异性免疫反应性抗原。在该测定的ELISPOT版本中，将细胞沉降到捕获抗体的固相表面上数小时，并且通过与检测抗体缀合的酶的沉淀产物检测所得的分泌和捕获的抗原。结果是在抗原分泌细胞的位点处出现有色斑点。该测定已被证明是非常灵敏的，因为它在释放后立即捕获分泌的蛋白质，并在扩散到更大的体积之前。该测定还被证明是独特的信息，因为它提供了关于细胞间变异的有价值的信息，这些信息在对来自细胞池的分泌蛋白进行的ELISA测定中丢失。 ELISPOT检测目前用于表征几种正常免疫功能和免疫细胞疾病，包括白血病、自身免疫性疾病和HIV/AIDS。ELISPOT测定的主要限制是它仅观察每个细胞的一种或最多两种分泌产物。Trellis以前开发了一种专有的基于细胞的多重显微镜技术，现在已将其扩展到ELISPOT，从而产生了真正的免疫斑点分析，CelISPOT。 第一阶段提案的具体目标是：1.使本手动系统自动化，用于阅读和分类CelISPOT细胞因子分泌模式; 2.将该测定从当前的5重鼠系统扩展至包括9种人细胞因子;以及3.使用该系统根据常规的Th 1和Th 2表型对人类T细胞进行分类，并确定单个细胞偏离这些类别的程度。 第一阶段工作的总体结果将是一个用于多重免疫斑点分析的全功能平台。在该项目的第二阶段，我们将把试剂集扩展到25种细胞因子，并与我们的外部合作者一起，我们将编制一个主要疾病适应症的结果数据库，以支持第三阶段的商业化。多重免疫斑点分析的可用性，为全面补充的细胞因子，具有足够的灵敏度，以检测罕见的细胞，预计将在临床医学中有深远的应用。CelISPOT检测的独特能力，详细表征病理表型将提供前所未有的精确度和灵敏度的表征免疫系统功能的广泛的重要疾病。