Development of novel assays for measuring concentration and metabolism of Tau in

描述（由申请人提供）：最近，博士。华盛顿大学医学院的Randall Bateman和David Holtzman开发了一种新的稳定同位素标记动力学（SILK）方法，用于测量人类中枢神经系统（CNS）中β淀粉样蛋白（AB）的代谢。该方法测量受试者输注稳定同位素标记的氨基酸后多个时间点标记AB与未标记AB的比例。在这种类型的研究中，当新的人类蛋白质被合成时，它们将加入标记的氨基酸。随着时间的推移，标记与未标记蛋白质的比例变化可以使用质谱仪测量，并可用于研究感兴趣的蛋白质的代谢。C2N已经成功地将这项技术商业化，目前正在美国国立卫生研究院（nih）资助的一项正在进行的研究中，扩大SILK检测，以涵盖多种AB亚型的生产和清除率。此外，C2N正在扩展SILK方法，使其包括稳定同位素峰值绝对定量（SISAQ），从而实现一种计算感兴趣蛋白质浓度的新方法。对于这项资助，我们建议扩展SILK和SISAQ方法，以包括脑脊液（CSF）中Tau蛋白的代谢和浓度的测量。SILK-Tau检测将主要用于新型Tau调节药物的临床试验。它将允许制药商在早期研究阶段评估他们的药物对人类大脑的影响。除了使用SILK测定Tau代谢外，我们认为SISAQ测定法具有互补和独立的价值。目前，测量生物体液中Tau浓度的唯一可用的分析方法是基于抗体的ELISA测定。已知通过ELISA测定的Tau浓度在实验室和研究之间差异很大，并且在位点内和位点之间都报告了相对较高的差异。根据我们对SISAQ- ab方法的初步经验，SISAQ检测的可变性明显低于我们在ELISA检测中看到的。该资助的第一阶段将侧重于开发方法，使我们能够使用SILK和SISAQ检测来测量Tau代谢和浓度。为了确认分析是有效的，我们将分析两名健康年轻受试者的全套CSF样本，这两名受试者已被招募参加正在进行的NIH SILK-AB同种异构体研究。该资助的II期将寻求进一步研究与II期临床研究相关的人群（轻度认知障碍和年龄匹配的对照组）中Tau的代谢。此外，我们将测试SISAQ-Tau测定的可重复性，并评估SISAQ-Tau和SISAQ-AB测定的组合作为潜在的诊断性生物标志物测定。

DESCRIPTION (provided by applicant): Recently, Drs. Randall Bateman and David Holtzman at the Washington University School of Medicine developed a novel stable isotope labeling kinetics (SILK) methodology for measuring the metabolism of amyloid beta (AB) in the human central nervous system (CNS). This methodology measures the ratio of labeled to unlabeled AB at multiple time points after subject infusion with a stable isotope labeled amino acid. In this tye of study, as new human proteins are synthesized, they will incorporate the labeled amino acid. The changes in the ratio of labeled to unlabeled protein over time can be measured using a mass spectrometer and can be used to investigate the metabolism of proteins of interest. C2N has successfully commercialized this technology and is currently expanding the SILK assay to encompass production and clearance rates of multiple AB isoforms in an ongoing NIH-funded study. In addition, C2N is expanding the SILK method to include stable isotope spike absolute quantitation (SISAQ), enabling a novel method for calculating the concentration of the protein of interest. For this grant, we propose to expand the SILK and SISAQ methodologies to include measurement of the metabolism and concentration of the protein Tau in cerebrospinal fluid (CSF). The SILK-Tau assay would be of prime interest for use in clinical trials of novel Tau modulating drugs. It would allow drug makers to assess the effect that their drugs have in the human brain in early stage studies. In addition to measuring metabolism of Tau using the SILK assay, we believe that the SISAQ assay has both complementary and standalone value. Presently, the only available analytical methods for measuring Tau concentrations in biological fluids are antibody based ELISA assays. Tau concentration measurements by ELISA are known to vary widely from lab to lab and from study to study with relatively high variances reported both within and between sites. Based on our preliminary experience with the SISAQ-AB method, the variability of the SISAQ assay is significantly lower than what we see in ELISA assays. Phase I of this grant will focus on development of methods that will allow us to measure Tau metabolism and concentration using the SILK and SISAQ assays. In order to confirm that the assays are working, we will analyze a full set of CSF samples from two of the healthy young subjects that have been recruited for the ongoing NIH SILK-AB isoform study. Phase II of this grant will seek to further investigate the metabolism of Tau in populations relevant to Phase II clinical studies (mild cognitive impairment and age matched controls). In addition, we will test the reproducibility of the SISAQ-Tau assay and assess the combination of the SISAQ-Tau and SISAQ-AB assay for use as a potential diagnostic biomarker assay. PUBLIC HEALTH RELEVANCE: C2N will develop a novel mass spectrometer based method for measuring the metabolism and concentration of Tau in human cerebrospinal fluid. We will use this method to assist pharmaceutical companies understand the effects that their Tau targeting drugs impart in the human brain. Tau is a rapidly emerging and attractive drug target for the prevention or delay of Alzheimer's disease progression.