SV40 滴定的快速方法。

Rapid method for SV40 titration.

机构信息

Department of Hematology, Hebrew University-Hadassah Medical School, Ein Kerem, Jerusalem, Israel.

出版信息

J Virol Methods. 2010 Mar;164(1-2):145-7. doi: 10.1016/j.jviromet.2009.12.003. Epub 2009 Dec 28.

DOI:10.1016/j.jviromet.2009.12.003

PMID:20005903

Abstract

SV40 titer is determined traditionally by the conventional plaque assay. Plaques appear after several rounds of infection and the assay takes around two weeks, which may delay research. A simpler assay was developed, based on detection of T-antigen in the infected cells by flow cytometry. Cells grown in 6-well plates are infected with serial dilutions of the viral stock, harvested 48h post-infection, stained and analyzed for T-antigen using a flow cytometer. The viral titer is calculated based on the percentage of T-antigen positive cells. The procedure is accomplished in 2 days. Unexpectedly we found that titers on different permissive African Green Monkey kidney cell lines were consistently different, suggesting variable susceptibility to SV40 infection. The method described, optimized for SV40 titration, may be adapted readily to other viruses.

摘要

SV40 滴度通常通过传统的空斑测定法来确定。空斑在几轮感染后出现，整个测定过程需要大约两周的时间，这可能会延迟研究进程。我们开发了一种更简单的测定法，该方法基于通过流式细胞术检测感染细胞中的 T 抗原。将细胞在 6 孔板中生长，用病毒储备液进行系列稀释后感染，在感染后 48 小时收获，使用流式细胞仪对 T 抗原进行染色和分析。根据 T 抗原阳性细胞的百分比计算病毒滴度。整个过程可在 2 天内完成。出人意料的是，我们发现不同的非洲绿猴肾细胞系的滴度始终存在差异，这表明对 SV40 感染的敏感性存在差异。本研究中描述的方法经过 SV40 滴定的优化，可轻松适用于其他病毒。