Multiplexed single cell cytokine secretion assay

描述(由申请人提供)：对正常免疫调节和疾病过程中的细胞因子网络的分析得益于免疫斑点分析的发展，该方法可以询问单个细胞是否分泌特定的免疫反应抗原。在ELISPOT版本的检测中，细胞停留在捕获抗体的固相表面上几个小时，所产生的分泌和捕获的抗原通过与检测器抗体结合的酶的沉淀产物进行检测。结果是在抗原分泌细胞的位置形成了一个彩色斑点。这种分析方法被证明是非常敏感的，因为它在释放后立即捕获分泌的蛋白质，然后扩散到更大的体积中。该分析还被证明具有独特的信息量，因为它提供了关于细胞间变异的有价值的信息，这些信息在对细胞池中分泌的蛋白质进行酶联免疫吸附试验时丢失。 ELISPOT试验目前被用来表征几种正常的免疫功能和免疫细胞疾病，包括白血病、自身免疫性疾病和艾滋病毒/艾滋病。ELISPOT分析的一个主要限制是每个细胞只能观察一个或最多两个分泌产物。Trellis之前开发了一种基于细胞的多路显微技术专利，现在将其扩展到ELISPOT，从而产生了一种真正的轮廓免疫斑点分析，CelISPOT。 这项第一阶段提案的具体目标是：1.自动化目前用于读取和分类CelISPOT细胞因子分泌模式的手动系统；2.将分析从目前的5-plex小鼠系统扩展到包括9种人类细胞因子；以及3.使用该系统根据传统的Th1和Th2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。 这项第一阶段工作的总体结果将是一个功能齐全的多重免疫斑点分析平台。在这个项目的第二阶段，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，编制一个重大疾病适应症结果的数据库，以支持第二阶段的商业化。多重免疫斑点分析可用于全面补充细胞因子，具有足够的灵敏度来检测稀有细胞，有望在临床医学中具有深远的应用。CelISPOT分析方法具有详细描述病理表型的独特能力，这将在描述多种重要疾病的免疫系统功能方面提供前所未有的精确度和灵敏度。

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.