ELISPOT FOR MEASUREMENT OF ANTIGEN SPECIFIC T CELLS

用于测量抗原特异性 T 细胞的 ELISPOT

• 批准号: 6563970
• 负责人: HERBERT KIM LYERLY
• 金额: $ 29.68万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-19 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6563970
• 关键词: T lymphocyte; active immunization; carcinoembryonal antigen; enzyme linked immunosorbent assay; genetically modified animals; laboratory mouse; neoplasm /cancer; neoplasm /cancer immunology; neoplasm /cancer immunotherapy; neoplasm /cancer vaccine; nonhuman therapy evaluation; tumor antigens

Description: (provided by applicant) Standard techniques used for the immunologic monitoring of cancer vaccine trials have included tritiated thymidine incorporation as a measure of antigen specific proliferation and the ability of cytotoxic T cells (CTL ) to lyse a tumor, most often assessed by chromium release. These two methods are difficult to reliably produce, require in vitro expansion, and are not quantitative. Addition of these assay systems to a limiting dilution analysis format has allowed a more accurate assessment of precursor frequency, but the number of immune effector cells needed to establish the analysis requires patients undergo a leukapheresis. Thus, these strategies are not viable for wide-scale immunologic monitoring. Antigen specific immune responses are largely regulated by secretion of cytokines whose function is to govern the growth and differentiation of T cell populations. Secretion of cytokines by T cells responding to a specific antigen has become a common measure of a functional immune response. Although the measurement of cytokine production by T cells using ELISA methodologies is not quantitative, measurement of cytokines offers a detailed characterization of the function of the T cell and the phenotype of the immune environment generated after antigen recognition. Recently, a single-cell assay has been developed to more accurately assess cytokine-producing cells. The method, termed ELIspot (enzyme-linked immunosorbant spot) can detect antigen reactive T cells before such cells could produce sufficient amount of protein to be detected by ELISA. Although ELIspot has become the "comparator" assay for immunologic monitoring, the sensitivity and limits of detection of the assay vary greatly from laboratory to laboratory . Obstacles to be overcome to translate ELIspot from a laboratory tool to a clinical grade monitoring technique include maximizing assay parameters to avoid any in vitro expansion step, develop the assay for use in cryopreserved cells, determine optimal antigen preparations used in analysis, and define the reliability of the assay to perform over time on the in multiple clinical samples. The specific aims of this proposal are to: (1) determine whether ELIspot can identify the T cell response to foreign protein antigens from the PBL of volunteer donors and cancer patients, (2) determine whether ELIspot can identify the T cell response to tumor antigens from the PBL of volunteer donors and cancer patients, (3) determine whether the T cell response, as measured by ELIspot, predicts tumor protection in murine transgenic tumor models such as the CEA and neu transgenic mice, and, finally, (4) determine whether ELIspot can predict immunization to cancer antigens and whether levels achieved are that similar to infectious disease antigens in human clinical trials of CEA, HER2, gp 100, and MAGE3 vaccines.

说明：（申请人提供） 用于癌症疫苗免疫学监测的标准技术 试验包括氚标记胸苷掺入作为抗原的测量 特异性增殖和细胞毒性T细胞（CTL）裂解A细胞的能力， 肿瘤，最常通过铬释放来评估。这两种方法 难以可靠地生产，需要体外扩增， 定量的在有限稀释分析中添加这些测定系统 格式允许对前兆频率进行更准确的评估，但 建立分析所需的免疫效应细胞的数量需要 患者接受白细胞去除术。因此，这些策略对于 大规模免疫监测。 抗原特异性免疫应答在很大程度上受免疫调节因子的分泌调节。 其功能是控制T细胞的生长和分化的细胞因子 人口。T细胞分泌细胞因子应答特异性 抗原已成为功能性免疫应答的常用量度。虽然 使用ELISA方法测量T细胞的细胞因子产生， 非定量的，细胞因子的测量提供了详细的表征 T细胞的功能和免疫环境的表型 抗原识别后生成。最近，一种单细胞测定法已经被应用于 以更准确地评估产生精氨酸的细胞。该方法， 称为ELIspot（酶联免疫吸附斑点）的方法可以检测抗原反应性 在这些细胞能够产生足够量的蛋白质之前， 通过ELISA检测。 尽管ELIspot已成为免疫学监测的“比较”试验， 该测定的灵敏度和检测极限从 从实验室到实验室翻译ELIspot需要克服的障碍 临床级监测技术的实验室工具包括最大化 测定参数为了避免任何体外扩增步骤，开发测定方法， 用于冻存细胞，确定用于 分析，并定义随着时间的推移在 在多个临床样本中。 该提案的具体目标是：（1）确定ELIspot是否可以 鉴定T细胞对来自PBL的外源蛋白抗原的应答， 志愿者捐赠者和癌症患者，（2）确定ELIspot是否可以 从志愿者PBL中鉴定T细胞对肿瘤抗原的应答 供体和癌症患者，（3）确定T细胞反应， 通过ELIspot测量，预测小鼠转基因肿瘤中的肿瘤保护作用 模型，如CEA和neu转基因小鼠，最后，（4）确定 ELIspot是否可以预测对癌抗原的免疫以及 获得了与人类临床上的传染病抗原相似的抗原， CEA、HER2、gp 100和MAGE 3疫苗的试验。