New low-viscosity overlay medium for viral plaque assays.
New low-viscosity overlay medium for viral plaque assays.
复制标题
用于病毒斑块测定的新的低粘度覆盖培养基。
DOI：
10.1186/1743-422x-3-63
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发表时间：
2006-08-31
期刊：
影响因子：
4.8
通讯作者：
Klenk HD
中科院分区：
文献类型：
--
作者：
Matrosovich M;Matrosovich T;Garten W;Klenk HD
固体或半固体覆盖介质下的单层细胞培养物中的空斑测定通常用于病毒和抗病毒物质的定量。为了克服已知覆盖的缺陷，我们测试了微晶纤维素 Avicel RC/CL™ 的悬浮液作为流感病毒空斑和空斑抑制测定中的覆盖介质。与琼脂和甲基纤维素 (MC) 覆盖介质相比，在含有 Avicel 的介质下形成了明显更大的噬菌斑。斑块尺寸随着 Avicel 浓度的降低而增加，但即使非常稀释的 Avicel 覆盖层 (0.3%) 也能确保局部斑块的形成。由于粘度低，Avicel 覆盖层比甲基纤维素覆盖层更容易使用，特别是在 96 孔培养板中。此外，可以在不事先去除病毒接种物的情况下应用 Avicel 覆盖层，从而有利于测定并减少交叉污染的机会。使用神经氨酸酶抑制剂奥司他韦羧酸盐，我们证明了基于 Avicel 的空斑减少测定法在抗病毒物质测试中的适用性。与琼脂和甲基纤维素覆盖层下的测定相比，在含有 Avicel 的覆盖介质下进行的噬菌斑测定更容易、更快且更灵敏。该测定可以很容易地在 96 孔板中进行，似乎特别适合高通量病毒滴定、血清学研究和病毒药物敏感性实验。它还可以促进在生物安全实验室的受限条件下进行高致病性病原体的工作。
Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CL™ as overlay media in the plaque and plaque-inhibition assay of influenza viruses. Significantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC) overlay media. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3%) ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances. Plaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs.