Masci Allyson L, Menesale Emily B, Chen Wei-Chiang, Co Carl, Lu Xiaohui, Bergelson Svetlana
Bioassay and Gene Therapy, Analytical Development, Biogen, Cambridge, MA, USA.
Mol Ther Methods Clin Dev. 2019 Jul 31;14:270-274. doi: 10.1016/j.omtm.2019.07.007. eCollection 2019 Sep 13.
Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation.
噬斑测定用于测量病毒样本的感染滴度。这些测定需要数天时间且通量较低，并且可能因人工噬斑计数存在偏差或主观性而受到分析人员差异的影响。通常，在第1天，将细胞过夜贴附于平板上。在第2天，用病毒感染细胞。再过3天后，固定噬斑，用辣根过氧化物酶（HRP）偶联抗体和HRP底物进行染色，然后人工计数。基于人工的噬斑视觉计数既耗时又费力，而且分析人员之间可能存在差异。此外，该测定必须进行数天，以使噬斑增大到足够大的尺寸以便人工识别。在此，我们整合荧光检测和自动噬斑计数以提高测定的灵敏度和速度。首先，我们用荧光标记抗体对噬斑进行染色。其次，我们使用基于平板的细胞成像仪进行无偏差、无主观性的噬斑计数。这两种技术的整合将测定时间缩短了40%，从5天缩短至3天，因为噬斑大小、噬斑信噪比和人工可视化不再是限制因素。这种优化的噬斑测定灵敏、快速且稳健，并扩大了该方法用于测量噬斑形成的通量和用途。
