Dee K U, Shuler M L
School of Chemical Engineering, Cornell University, Ithaca, New York 14853, USA.
Biotechnol Prog. 1997 Jan-Feb;13(1):14-24. doi: 10.1021/bp960086t.
We have previously described a quantitative model of the trafficking of baculovirus in insect cells that considers the various infection steps such as attachment, internalization, endosomal fusion, and nuclear accumulation. Concepts from the model were used to design synchronous infection regimens for various cell lines, to analyze the inherent inefficiency of existing assays for virus titer, and to develop a modified end-point dilution assay optimized to measure more completely the concentration of intrinsically infectious particles in a virus inoculum. The titer obtained from existing assays incompletely counts infectious virus particles due primarily to the incomplete adsorption of the virus during the short, standard 1-h incubation period. For representative assays, the calculated bound virus is generally about 10% of the added virus, but could be as low as 1.4%, underestimating actual titers by 3-70-fold. A modified end-point dilution assay involving centrifugation has been developed from both quantitative and qualitative analyses. The ratio of particle to plaque-forming unit with the optimized assay was 4-6 compared to 100-300 for typical assays, representing a significant improvement in the measurement of total infectious virus concentration.
我们之前描述了杆状病毒在昆虫细胞中运输的定量模型，该模型考虑了诸如附着、内化、内体融合和核积累等各种感染步骤。该模型中的概念被用于设计针对各种细胞系的同步感染方案，分析现有病毒滴度检测方法固有的低效性，并开发一种改良的终点稀释检测方法，该方法经过优化，能更全面地测量病毒接种物中固有感染性颗粒的浓度。现有检测方法获得的滴度不能完全计数感染性病毒颗粒，主要原因是在标准的1小时短孵育期内病毒吸附不完全。对于代表性检测方法，计算得出的结合病毒通常约为添加病毒的10%，但可能低至1.4%，实际滴度被低估3至70倍。通过定量和定性分析开发了一种改良的终点稀释检测方法，该方法涉及离心。优化后的检测方法中颗粒与噬斑形成单位的比率为4至6，而典型检测方法为100至300，这表明在测量总感染性病毒浓度方面有了显著改进。
