PDF(Pubmed)
Abstract：
Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.
摘要：
感染性测定是开发和制造基于病毒的治疗剂的关键分析技术。这里,我们介绍了一种新的分析格式,该格式利用无标记亮场图像来确定细胞形态中感染依赖性变化的动力学.特别是,细胞舍入与所施用的感染性病毒的量成正比，能够相对于标准曲线快速测定病毒滴度。我们的动力学感染性病毒滴度(KIT)测定是稳定性指示，由于其灵敏的读出方法，提供感染后24小时内的结果。与传统的感染性检测相比，这取决于感染终点的单个读数，基于每个样品仅三个孔,通过拟合模型对动力学数据进行累积分析得到精确的结果(CV<20%)。这种方法允许通过单个操作员每周处理约400个样品的高通量。我们证明了KIT测定对基因工程溶瘤VSV-GP的适用性，新城疫病毒（NDV），副痘病毒(ORFV)但它可能会扩展到广泛的病毒，这些病毒在感染后会引起形态变化。这种测定法的多功能性，结合其独立于特定仪器或软件，使其成为克服制药行业感染性测定分析瓶颈的有希望的解决方案，并作为学术研究的常规方法。
