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

 描述（由申请方提供）：本提案是对RFA-A1-14-027：“用于监测免疫应答的样品保留试验的开发”（U24）的回应。我们将开发20种细胞因子的超灵敏检测方法，这些细胞因子在过去的血清和血浆中很大程度上无法测量。将在全自动仪器上以用户定义的多重组合进行测定，灵敏度为个位数fg/mL，每次测量一组10种分析物（包括仪器死体积）时仅使用55 uL样品。在分析验证系统后，我们将与学术团体合作， NIAID DAIT网络。 我们将通过两种方式实现样品节约：第一种是多路复用（每孔10次检测），这将每次检测的样品使用量减少了一个数量级;第二种是采用一种新型的超灵敏检测技术，将检测灵敏度提高了约100倍。我们已经证明了使用该技术的几种细胞因子测定的检测限为lfg/mL或更好（Glezer等人，2014）。这种增加的灵敏度可用于测量样品中的细胞因子，其中水平太低而无法使用当前技术检测。对于许多应用，单位数fg/mL的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。 开发的检测方法将在超过1，000台MSD仪器的大型安装基础上提供，并提供手动检测方案。此外，这些检测将在全自动分析仪上进行，这不仅为研究界提供了方便、节省劳动力的工具，而且还提高了检测的重现性和鲁棒性。 在该项目的前两年，我们将开发16种超灵敏的细胞因子检测方法，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路复用。在第四年，这20种超灵敏检测方法将被转移到一个自动化平台（在独立资助下开发）。在最后一年，将进行分析性能验证研究和外部验证研究。

 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.