Shen Chun Fang, Meghrous Jamal, Kamen Amine
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave, Montréal, Quebec, Canada H4P 2R2.
J Virol Methods. 2002 Sep;105(2):321-30. doi: 10.1016/s0166-0934(02)00128-3.
A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.
开发了一种使用流式细胞术（FCM）分析来定量昆虫细胞培养物中产生的杆状病毒总颗粒的方法。该方法是对颗粒进行直接计数，涉及用SYBR Green I（一种高荧光核酸特异性染料）对杆状病毒DNA进行染色。含有出芽病毒颗粒的无细胞上清液的样品制备包括用多聚甲醛固定、冻融处理、用Triton X-100使病毒膜通透化以及加热样品以提高染色效率并增强杆状病毒颗粒的绿色荧光强度。在本研究中，研究了不同处理步骤和培养基组成对病毒颗粒计数的影响，以确定最佳制备条件。在病毒浓度范围为两个对数的情况下建立了FCM分析的线性，检测下限为每毫升10(5)个病毒颗粒。使用来自大规模生物反应器培养物的样品评估了该方法的稳健性和可重复性。通过FCM分析获得的事件（或病毒颗粒计数）通常高于通过终点稀释法（EPDA）获得的滴度。来自16种不同病毒株的结果显示，总颗粒（FCM）与感染性颗粒（EPDA）的平均比例为3.7。基本上，以下报道的FCM分析将杆状病毒定量时间缩短至2小时，并能很好地估计病毒滴度。据信，这些发现将有助于加速杆状病毒表达技术领域的工艺开发，特别是在细胞的化学计量多病毒感染对复杂产物表达至关重要的工艺中。
