病毒滴度测定的新兴及改良方法进展

doi:10.19586/j.2095-2341.2024.0026

生物技术进展 ›› 2024, Vol. 14 ›› Issue (3): 377-387.DOI: 10.19586/j.2095-2341.2024.0026

• 进展评述 • 上一篇

病毒滴度测定的新兴及改良方法进展

杜丽萍(), 徐明明(), 官艳东, 周沙沙, 张杰

北京义翘神州科技股份有限公司检测部，北京 100176

收稿日期:2024-02-23 接受日期:2024-03-21 出版日期:2024-05-25 发布日期:2024-06-18
通讯作者: 徐明明
作者简介:杜丽萍 E-mail: 15774608960@163.com；

Advances in Emerging and Improved Methods for Detecting Viral Titers

Liping DU(), Mingming XU(), Yandong GUAN, Shasha ZHOU, Jie ZHANG

Testing Department of Sino Biological Inc. ，Beijing 100176，China

Received:2024-02-23 Accepted:2024-03-21 Online:2024-05-25 Published:2024-06-18
Contact: Mingming XU

摘要/Abstract

摘要：

病毒滴度测定是生物制药行业重要的分析方法，广泛应用于病毒类生物制品的开发和生产、病毒清除灭活工艺验证、外源病毒检测等领域，以确保病毒类生物制剂的活性和有效性，以及生物制品的病毒安全性。因此，建立快速、简单且准确的病毒滴定检测方法尤为重要。总结了检测病毒滴度的传统方法、新兴以及改良方法的特点、原理及具体应用，并比较了各自的优缺点。一些新兴方法，如微滴式数字PCR、病毒定量毛细管电泳、化学发光ISH-PNA测定、激光力细胞学等改进了传统方法耗时耗力、重复性差、准确性低、结果主观性大的缺点，达到了快速、灵敏、自动化程度高、精密度高、结果更加稳健且客观的优点，但部分新方法仪器昂贵或者未广泛使用，需要根据实验目的选择合适的病毒滴定方法。

关键词: 生物制药, 病毒清除/灭活验证, 病毒感染性, 病毒滴定

Abstract:

Virus titration is an important analytical method in the biopharmaceutical industry， which is widely used in the development and production of viral biological products， the validation of virus clearance and inactivation processes， and the detection of exogenous viruses to ensure the activity and effectiveness of viral biologics， as well as the viral safety of biological products. It is important to establish a fast， simple， and accurate method for the detection of virus titration. This article summarized the characteristics， principles and specific applications of the traditional， emerging and improved methods for detecting viral titer and compared their advantages and disadvantages. Some emerging methods， such as droplet digital PCR， viral quantitative capillary electrophoresis， in situ hybridization （ISH） assay with chemiluminescent detection， laser force cytology， etc.， have improved the shortcomings of traditional methods such as time-consuming and labor-intensive， poor repeatability， low accuracy， and large subjectivity of results， and have achieved the advantages of rapidity， sensitivity， high degree of automation， high precision， and more robust and objective results. However， some new methods are expensive or not widely used， and it is necessary to select the appropriate virus titration method according to the experimental purpose.

Key words: biopharmaceutical, viral clearance/inactivation validation, viral infectivity, virus titration

中图分类号:

Q93-33

杜丽萍, 徐明明, 官艳东, 周沙沙, 张杰. 病毒滴度测定的新兴及改良方法进展[J]. 生物技术进展, 2024, 14(3): 377-387.

Liping DU, Mingming XU, Yandong GUAN, Shasha ZHOU, Jie ZHANG. Advances in Emerging and Improved Methods for Detecting Viral Titers[J]. Current Biotechnology, 2024, 14(3): 377-387.

图/表 1

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方法分类	方法名称	时间成本	费用成本	使用仪器	适用病毒种类	优点	缺点	可操作性
病毒感染水平	PFU法	天~周	*	肉眼或低倍显微镜	产生CPE的病毒	操作简单	重复性差，费时费力，自动化程度差，受主观影响性大，需要病毒在培养时有复制周期	操作简单，工作量大，需要有经验的检测人员
	TCID₅₀法	天~周	*	普通显微镜	产生CPE的病毒	操作简单	耗时耗力，需要重复多次	操作简单，工作量大，需要有经验的检测人员
	荧光灶试验	天	**	荧光显微镜	需要特异性病毒抗体	重复性高、降低主观性	需要特异性病毒抗体	操作较简单，通过预实验确定特异性抗体使用浓度，熟练操作荧光显微镜
	IPMA法	天	**	荧光显微镜	需要特异性病毒抗体	高灵敏度、重复性和特异性好	结果受个别主观因素影响，且病毒含量过低时无法产生特定的颜色反应，样品阳性检出率略低	操作简单，方法成熟，实验所需试剂和耗材成本低
病毒蛋白水平	ELISA	小时~天	**	酶标仪	广泛应用	省时	严格性低，测量的病毒元素不一定与病毒颗粒有关	操作较简单，通过预实验确定相关实验细节
病毒蛋白水平	病毒定量毛细管电泳	小时~天	**	毛细管	广泛应用	样品使用量小、分辨率高、操作简单	主要应用在病毒类疫苗的质量控制，不是严格意义上的病毒滴定	操作简单
病毒核酸水平	qPCR	小时	*	qPCR仪	广泛应用	快速、灵敏、稳定	不能确保仅对感染颗粒进行定量	操作简单，考验操作人员精细程度
	ddPCR	小时	**	微液滴数字PCR仪	广泛应用	准确度和精密度高，无需标准曲线或特殊的样品制备，可对目标基因实现绝对定量	通量限制，花费大	操作简单，但受通量限制，工作量较大
	产物增强逆转录酶测定	小时~天	**	qPCR仪	主要应用于逆转录病毒，如异种鼠白血病病毒和C型CHO细胞颗粒中所含的RT活性	特异性、准确性、精密度高、稳定	仅适用于逆转录病毒类	操作简单
	化学发光ISH-PNA测定	分~小时	***	荧光显微镜+ CCD相机	B19细小病毒	高分辨率，可在单细胞内提供病毒核酸的清晰定位，准确作出病毒感染周期后感染细胞百分比的客观评估，在单细胞水平上对病毒核酸进行定量	前期耗材多，需要生物素标记的PNA探针，在其他病毒上是否可行有待验证	操作较简单，通过预实验确定特异性抗体使用浓度
病毒颗粒水平	OD₂₆₀	分	*	分光光度计	纯化后病毒	快速、简单	不能区分有感染力和无感染力病毒，病毒需纯化	操作简单
	FACS	分~小时	***	流式细胞仪	病毒需有荧光标记或使用特异性病毒抗体染色	快速、可靠	仪器花费大，标记病毒蛋白的荧光抗体偶联物试剂不容易获得	熟练掌握流式细胞仪操作，通过预实验确定特异性病毒抗体使用浓度
	TEM	小时	****	电子显微镜	广泛应用	可靠、直观	有细胞衍生颗粒的污染（如外泌体），通量低，精确定量难度大	操作复杂，需熟练掌握负染色技术和电镜技术
	激光力细胞学	小时~天	****	Radiance LFC 仪	广泛应用	省时、操作简单	仪器昂贵	仪器不常见，对检测人员要求高

方法分类	方法名称	时间成本	费用成本	使用仪器	适用病毒种类	优点	缺点	可操作性
病毒感染水平	PFU法	天~周	*	肉眼或低倍显微镜	产生CPE的病毒	操作简单	重复性差，费时费力，自动化程度差，受主观影响性大，需要病毒在培养时有复制周期	操作简单，工作量大，需要有经验的检测人员
	TCID₅₀法	天~周	*	普通显微镜	产生CPE的病毒	操作简单	耗时耗力，需要重复多次	操作简单，工作量大，需要有经验的检测人员
	荧光灶试验	天	**	荧光显微镜	需要特异性病毒抗体	重复性高、降低主观性	需要特异性病毒抗体	操作较简单，通过预实验确定特异性抗体使用浓度，熟练操作荧光显微镜
	IPMA法	天	**	荧光显微镜	需要特异性病毒抗体	高灵敏度、重复性和特异性好	结果受个别主观因素影响，且病毒含量过低时无法产生特定的颜色反应，样品阳性检出率略低	操作简单，方法成熟，实验所需试剂和耗材成本低
病毒蛋白水平	ELISA	小时~天	**	酶标仪	广泛应用	省时	严格性低，测量的病毒元素不一定与病毒颗粒有关	操作较简单，通过预实验确定相关实验细节
病毒蛋白水平	病毒定量毛细管电泳	小时~天	**	毛细管	广泛应用	样品使用量小、分辨率高、操作简单	主要应用在病毒类疫苗的质量控制，不是严格意义上的病毒滴定	操作简单
病毒核酸水平	qPCR	小时	*	qPCR仪	广泛应用	快速、灵敏、稳定	不能确保仅对感染颗粒进行定量	操作简单，考验操作人员精细程度
	ddPCR	小时	**	微液滴数字PCR仪	广泛应用	准确度和精密度高，无需标准曲线或特殊的样品制备，可对目标基因实现绝对定量	通量限制，花费大	操作简单，但受通量限制，工作量较大
	产物增强逆转录酶测定	小时~天	**	qPCR仪	主要应用于逆转录病毒，如异种鼠白血病病毒和C型CHO细胞颗粒中所含的RT活性	特异性、准确性、精密度高、稳定	仅适用于逆转录病毒类	操作简单
	化学发光ISH-PNA测定	分~小时	***	荧光显微镜+ CCD相机	B19细小病毒	高分辨率，可在单细胞内提供病毒核酸的清晰定位，准确作出病毒感染周期后感染细胞百分比的客观评估，在单细胞水平上对病毒核酸进行定量	前期耗材多，需要生物素标记的PNA探针，在其他病毒上是否可行有待验证	操作较简单，通过预实验确定特异性抗体使用浓度
病毒颗粒水平	OD₂₆₀	分	*	分光光度计	纯化后病毒	快速、简单	不能区分有感染力和无感染力病毒，病毒需纯化	操作简单
	FACS	分~小时	***	流式细胞仪	病毒需有荧光标记或使用特异性病毒抗体染色	快速、可靠	仪器花费大，标记病毒蛋白的荧光抗体偶联物试剂不容易获得	熟练掌握流式细胞仪操作，通过预实验确定特异性病毒抗体使用浓度
	TEM	小时	****	电子显微镜	广泛应用	可靠、直观	有细胞衍生颗粒的污染（如外泌体），通量低，精确定量难度大	操作复杂，需熟练掌握负染色技术和电镜技术
	激光力细胞学	小时~天	****	Radiance LFC 仪	广泛应用	省时、操作简单	仪器昂贵	仪器不常见，对检测人员要求高

[1]	张永红, 李岩异, 刘容麟, 吕娜, 张卫婷. IgY在抗病毒医学领域中的应用[J]. 生物技术进展, 2024, 14(1): 35-41.
[2]	杜力,刘晓志,魏敬双,高健. 蛋白质药物糖基化工程化改造研究进展[J]. 生物技术进展, 2020, 10(5): 448-455.

病毒滴度测定的新兴及改良方法进展

Advances in Emerging and Improved Methods for Detecting Viral Titers

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