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

 描述(由申请人提供)：本建议是对RFA-A1-14-027：“建立用于监测免疫反应的样本保存分析”(U24)的回应。我们将开发对过去在血清和血浆中基本上无法测量的20种细胞因子的超灵敏分析。分析将在全自动仪器上以用户定义的多路组合进行，灵敏度为个位数Fg/mL，每次测量10个分析物(包括仪器死体积)时仅使用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在 NIAID DAIT网络。 我们将通过两种方法实现样本节省：第一，通过多路传输(每孔10次分析)，将每次测试的样本使用量减少一个数量级；第二，使用一种新的超灵敏测试技术，将测试灵敏度提高约100倍。我们已经证明，使用这项技术进行几种细胞因子分析的检测下限为1fg/mL或更高(Glezer等人，2014年)。这种提高的灵敏度可以用来测量样本中的细胞因子，这些样本的水平太低，使用目前的技术无法检测到。对于许多应用，个位数的fg/m L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。 开发的分析将在1000多台MSD仪器的大型安装基础上使用，并具有手动分析协议。此外，分析将在全自动分析仪上使用，这不仅为研究团体提供了一个方便、省力的工具，而且还提高了分析的重复性和稳健性。 在这个项目的头两年，我们将开发16种超灵敏的细胞因子检测方法，检测下限为10fg/ml或更高(另外四种检测方法已经可用)。在第三年，我们将演示手动格式的多路复用。在第四年，这20种超灵敏分析将被转移到一个自动化平台(由独立资金开发)。在去年，将进行一项分析性业绩核查研究和一项外部验证研究。

 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.