Svensson L, Hjalmarsson A, Everitt E
Department of Microbiology, Lund University, Sweden.
J Virol Methods. 1999 Jun;80(1):17-24. doi: 10.1016/s0166-0934(99)00018-x.
Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4-8 degrees C for 2 months in the salt buffer used commonly.
一些细胞系难以在汇合的单层中生长，因此，由于蚀斑可能难以与细胞单层的其他空白区域区分开来，所以不能应用蚀斑测定法。为避免这个问题，开发了一种快速灵敏的免疫斑点印迹TCID50方法，使用针对病毒抗原的抗体来检测感染和病毒产生。开发了一种替代统计方法来处理评分数据，从而获得了10%的变异系数。为了加快整个过程并提高在96孔细胞培养板中生长的细胞的增殖率，将培养板在4℃下用从含有汇合细胞单层的细胞培养瓶中获得的生长培养基预处理4小时。这种免疫斑点印迹TCID50方法被用于研究传染性胰腺坏死病毒（IPNV）储存后的感染性维持情况。在-70℃下用冷冻保护剂（10%甘油）储存IPNV，即使经过多达十次的冻融循环，仍能保持TCID50水平，而在常用的盐缓冲液中于4-8℃储存2个月后，感染性下降了四个数量级。
