疾病相关生物标志物的测量已成为临床试验设计的主要组成部分,但是在缺乏严格的临床和分析验证检测方法的情况下,对结果的解释可能会产生误导。亨廷顿病(HD),测定患者脑脊液(CSF)中突变型亨廷顿蛋白(mHTT)的浓度可作为降低HTT治疗方法的疾病进展生物标志物和药效学读数.我们最近通过一种新颖的基于超灵敏免疫测定的技术发表了HD患者CSF中mHTT水平的定量,并在此对其进行了分析验证。
这项工作旨在分析和临床验证我们对人类HDCSF中mHTT测量的超灵敏测定,作为CNSmHTT降低的药效学生物标志物在临床试验中的应用。
单分子计数(SMC)测定是一种基于珠子的超灵敏免疫测定,染料标记的抗体被共聚焦激光激发并发射荧光作为读数。根据已建立的食品和药物管理局和欧洲药品管理局指南,对该技术对mHTT的检测进行了临床验证。
SMC分析被证明是准确的,精确,具体,和可重复的。虽然没有检测到基质影响,我们编制了一份干扰物质清单,作为正确收集和储存患者CSF样本的指南.此外,提供了一套关于结果解释的建议。
该SMC测定是用于人CSF中mHTT的相对定量的稳健且超灵敏的方法。
The measurement of disease-relevant biomarkers has become a major component of clinical trial design, but in the absence of rigorous clinical and analytical validation of detection methodology, interpretation of results may be misleading. In Huntington\'s disease (HD), measurement of the concentration of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technology and here analytically validate it for use.
This work aims to analytically and clinically validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clinical trials.
The single molecule counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technology was clinically validated following established Food and Drug Administration and European Medicine Agency guidelines.
The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addition, a set of recommendations on result interpretation is provided.
This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.