Opening a window into immune system signaling at the fg/ml level

在 fg/ml 水平上打开免疫系统信号传导的窗口

• 批准号: 9099778
• 负责人: Martin Stengelin
• 金额: $ 39.67万
• 依托单位: MESO SCALE DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-26 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099778
• 关键词: Automation; Biological Assay; Blood; Clinical Research; Collaborations; Communities; Confidential Information; Custom; Detection; Development; Digit structure; Disease; Drops; Evaluation; Funding; Generations; Government; Health; Hour; Human; Immune response; Immune system; Manuals; Measurable; Measurement; Measures; Medical; Methods; Monitor; National Institute of Allergy and Infectious Disease; Oligonucleotides; Performance; Persons; Plasma; Plasma Cells; Protocols documentation; Recovery; Reproducibility; Research; Research Personnel; Role; Running; Sampling; Serum; Signal Transduction; Signaling Protein; Spottings; Staging; System; Technology; Testing; Time; Tissues; Validation; base; cross reactivity; cytokine; improved; instrument; meetings; new technology; novel; response; tool; validation studies

 DESCRIPTION (provided by applicant): This proposal is in response to RFA-A1-14-027: "Development of Sample Sparing Assays for Monitoring Immune Responses" (U24). We will develop ultrasensitive assays for 20 cytokines that have largely been unmeasurable in serum and plasma in the past. The assays will be carried out on a fully automated instrument in user-defined multiplexed combinations, at single-digit fg/mL sensitivity, using only 55 uL of sample per measurement of a set of ten analytes (instrument dead volume included). After analytically validating the system, we will demonstrate its utility in collaboration with academic groups in the NIAID DAIT network. We will implement sample sparing by two means: first by multiplexing (ten assays per well), which reduces sample use per assay by an order of magnitude; second, by employing a novel ultrasensitive assay technology that improves assay sensitivity approximately 100-fold. We have demonstrated detection limits of 1 fg/mL or better for several cytokine assays using this technology (Glezer et al., 2014). This increased sensitivity can be used to measure cytokines in samples where levels are too low to be detectable using current technologies. For many applications, the single-digit fg/mL sensitivity will allow the sample to be diluted, resulting in further reduction in the volume of sample required. The developed assays will be available on the large installed base of over 1,000 MSD instruments, with manual assay protocols. Additionally, the assays will be available on a fully automated analyzer, which not only provides the research community with a convenient, labor saving tool, but by also improves reproducibility and robustness of the assays. In the first two years of this project, we will develop 16 ultrasensitive cytokine assays with detection limits of 10 fg/mL or better (an additional four assays are already available). In year three, we will demonstrate multiplexing in a manual format. In year four, these 20 ultrasensitive assays will be transferred to an automated platform (developed under independent funding). In the last year, an analytical performance verification study and an external validation study will be performed.

 描述（由申请人提供）：本提案是对 RFA-A1-14-027 的回应：“开发用于监测免疫反应的样品保留测定法”(U24) 我们将为 20 种细胞因子开发超灵敏测定法，这些细胞因子在很大程度上无法测量。过去，这些检测将在全自动仪器上以用户定义的多重组合进行，浓度为单位数 fg/mL。灵敏度，每次测量一组十种分析物（包括仪器死体积）仅使用 55 uL 样品。在对系统进行分析验证后，我们将与学术团体合作展示其实用性。 NIAID DAIT 网络。 我们将通过两种方式实现样品节省：首先是通过多重检测（每孔数十个），这可以将每次检测的检测样本使用量减少一个数量级；其次是采用新型超灵敏检测技术，将检测灵敏度提高约 100 倍。使用该技术的多种细胞因子测定的检测限为 1 fg/mL 或更好（Glezer 等人，2014），这种提高的灵敏度可用于测量水平太低而无法检测到的样品中的细胞因子。对于许多应用，单位数的 fg/mL 灵敏度将允许样品被稀释，从而进一步减少所需的样品体积。 所开发的检测方法将在超过 1,000 台 MSD 仪器的大型安装基础上使用，并具有手动检测方案。此外，这些检测方法将在全自动分析仪上使用，这不仅为研究界提供了一种方便、省力的工具，而且还提高了测定的再现性和稳健性。 在该项目的前两年，我们将开发 16 种超灵敏细胞因子检测方法，检测限为 10 fg/mL 或更高（另外四种检测方法已经可用）。在第三年，我们将以手动格式演示多重检测。第四，这20个超灵敏检测将转移到自动化平台（在独立资助下开发），去年将进行分析性能验证研究和外部验证研究。