An immunological assay for determination of baculovirus titers in 48 hours
An immunological assay for determination of baculovirus titers in 48 hours
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DOI：
10.1006/abio.1998.3042
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发表时间：
1999-03-15
影响因子：
2.9
通讯作者：
Green, G
中科院分区：
文献类型：
--
作者：
Kitts, PA;Green, G
杆状病毒表达系统是表达真核蛋白的首选系统。用表达目标蛋白的杆状病毒感染昆虫细胞，可以产生大量的生物活性物质。在杆状病毒砧木生产的几个阶段，有必要对病毒滴度进行检测。目前的方法耗时很长，要么技术上困难，要么仅限于表达报告基因的病毒。本文描述的新检测方法在48小时内产生滴度，易于使用96孔板进行检测，并且适用于任何基于加州签名虫核多角体病毒的重组杆状病毒。该试验使用病毒包膜糖蛋白抗体，通过免疫染色检测感染细胞。滴度是通过在光镜下计数感染灶来确定的。所需的潜伏期大大缩短，因为感染的细胞表达病毒抗原早在其他检测中感染的宏观迹象变得明显之前。使用该免疫测定法测定的滴度与使用传统方法获得的滴度在数值和变异性方面具有可比性，前提是该样品的滴度高于10(4)pfu/ml。(C) 1999学术出版社。
Th baculovirus expression system is a system of choice for expressing eukaryotic proteins. Large amounts of biologically active material can be generated using this system by infecting insect cells with a baculovirus expressing the target protein. At several stages during the production of a baculovirus stock, it is necessary to titer the virus. Current methods have long time lines and are either technically difficult or are limited to viruses expressing a reporter gene. The new assay described here yields titers in 48 h, is easy to perform using 96-well plates, and is applicable to any Autographa californica nucleopolyhedrovirus-based recombinant baculovirus. This assay uses an antibody to a viral envelope glycoprotein to detect infected cells via immunostaining. The titer is determined by counting foci of infection under a light microscope. The required incubation period is shortened considerably because infected cells express viral antigens long before the macroscopic signs of infection scored in other assays become apparent. Titers determined using this immunological assay are comparable, both in value and variability, to those obtained using a traditional method, provided that the stocks have titers above 10(4) pfu/ml. (C) 1999 Academic Press.