A Novel Influenza Neuraminidase Inhibition Assay

描述（由申请方提供）：I期SBIR项目的主要目标是确定新型神经氨酸酶检测试剂盒用作流感神经氨酸酶抑制和药物敏感性检测试剂盒的可行性。所提出的测定法是一种生物化学发光测定法，其使用新型底物并允许神经氨酸酶活性的真实的时间测定，其基本上仅需要一个制备步骤-将样品添加到含有测定法所需的所有试剂的主检测混合物中。建议的努力包括1）改进底物合成，2）评估作为NI和药物敏感性测定的可行性，以及3）确定该测定是否具有足够的灵敏度，以便临床标本可以直接用于该测定。两种神经氨酸酶抑制剂-扎那米韦，特别是奥司他韦-是目前在流感流行和大流行期间药物干预的支柱。抗药性流感病毒变异体的出现加剧了人们对这些药物广泛使用的担忧。必须监测抗病毒药物耐药变异的出现。基于细胞和生物化学的测定已用于确定病毒对神经氨酸酶抑制剂的易感性。细胞测定具有许多缺点，包括在细胞培养期间补偿神经氨酸酶抑制剂抗性的突变的选择、长的测定时间和繁琐的程序，这使得它们不太适合于大规模监测程序。相比之下，生化测定直接测量病毒神经氨酸酶活性的抑制，从而规避了与细胞测定相关的许多问题。然而，目前的生化测定仍然很复杂，需要数小时才能完成，并且具有显著的测定可变性。更重要的是，它们缺乏足够的灵敏度以允许直接使用临床样本（例如，洗鼻剂），这是大规模监测程序的更优选的样品类型。成功开发拟议的检测试剂盒将允许使用临床标本进行药物敏感性检测-这是对目前使用的检测试剂盒的重大改进，目前使用的检测试剂盒必须使用通过冗长繁琐的培养方法获得的病毒分离株。公共卫生相关性：两种神经氨酸酶抑制剂-扎那米韦和奥司他韦-是目前流感流行或大流行期间药物干预的主要药物。鉴于耐药流感病毒变异体出现的历史，必须监测抗病毒耐药变异体的出现，这需要稳健的神经氨酸酶抑制（NI）测定。建议的努力是确定一种新的神经氨酸酶活性测定作为流感NI测定的可行性。具体而言，我们将确定该检测方法是否1）快速（5分钟制备时间加30分钟孵育）; 2）简单（基本上是一步操作）; 3）灵敏（相当于或优于25个循环的真实的PCR检测），临床标本可直接用于NI检测; 4）重复性好（CV<15%）;和5）适用于大规模样品检测的96孔格式。

DESCRIPTION (provided by applicant): The primary goal of the Phase I SBIR projects is to determine the feasibility of a novel neuraminidase assay to be used as an influenza neuraminidase inhibition and drug susceptibility assay. The proposed assay is a biochemiluminescence assay that uses a novel substrate and permits real time determination of neuraminidase activity, which essentially requires only one preparation step - the addition of sample to a master detection mix that contains all necessary reagents for the assay. The proposed efforts include 1) improving substrate synthesis, 2) evaluating the feasibility as a NI and drug susceptibility assay, and 3) determining whether the assay has sufficient sensitivity so that clinical specimens can be directly used for the assay. Two neuraminidase inhibitors - zanamivir and particularly oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic and, if happened, a pandemic. Emergence of drug resistant influenza virus variants heightens the concerns about widespread use of these drugs. It is imperative to monitor the emergence of antiviral drug resistant variants. Both cellular and biochemical based assays have been used to determine viral susceptibility to neuraminidase inhibitors. Cellular assays suffer from a number of drawbacks, including selection of mutations that compensate for neuraminidase inhibitor resistance during cell culture, long assay time, and tedious procedures, which make them less suitable for large scale surveillance programs. In contrast, the biochemical assays directly measure the inhibition of viral neuraminidase activities, thus circumventing many of the problems associated with the cellular assays. However, current biochemical assays are still complicated, take hours to complete, and have significant assay variability. More importantly, they lack sufficient sensitivity to allow drug susceptibility determination directly using clinical specimens (e.g., nasal wash), a much preferred sample type for large scale surveillance programs. Successful development of the proposed assay will allow the use of clinical specimens for drug susceptibility testing - a significant improvement over the currently used assays, which have to use virus isolates that are obtained via the lengthy and cumbersome culture methods. PUBLIC HEALTH RELEVANCE: Two neuraminidase inhibitors - zanamivir and oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic or pandemic. Given the history of emergence of drug resistant influenza virus variants, it is imperative to monitor the emergence of antiviral drug resistant variants, which requires a robust neuraminidase inhibition (NI) assay. The proposed efforts are to determine the feasibility of a novel neuraminidase activity assay as an influenza NI assay. Specifically, we will determine if the assay is 1) rapid (5 min preparation time plus 30 min incubation); 2) simple (essentially one step operation); 3) sensitive (equivalent to or better than 25 cycles of real time PCR assay) so that clinical specimens can be directly used for NI assay; 4) reproducible (CV<15%); and 5) suitable for a 96-well format for large-scale sample detection.