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试验的发展，该试验可以询问单个细胞分泌特异性免疫反应性抗原。在ELISPOT版本中，将细胞沉淀在捕获抗体的固相表面上数小时，通过与检测器抗体偶联的酶的沉淀产物检测产生的分泌和捕获的抗原。结果是在抗原分泌细胞的位置有一个彩色的斑点。这种检测方法被证明是非常敏感的，因为它在释放后和扩散到更大体积之前立即捕获分泌的蛋白质。该分析也被证明具有独特的信息性，因为它提供了关于细胞间变异的有价值的信息，而这些信息是在ELISA分析中对细胞池分泌的蛋白质进行分析时丢失的。

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.