Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO4 as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.
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机译：蔗糖密度梯度的超速离心仍然是hRSV纯化最常用的技术。然而，蔗糖的高粘度和高渗透性会导致对极不稳定的病毒的损害，从而导致传染性的丧失。为了克服这些局限性，开发了一种替代的纯化技术，使用碘克沙醇作为梯度培养基，在感染性测定之前加入了硫酸镁作为稳定剂和EDTA来分解病毒。通过在20％至52％的碘克沙醇不连续梯度上进行速率区域超速离心纯化聚乙二醇浓缩的病毒后，病毒颗粒在20％至36％的界面处结合。使用ELISA通过病毒融合糖蛋白含量证实了病毒的存在。通过在20-52％的连续梯度上通过浮力密度超速离心进一步纯化后，病毒在密度为1.15-1.19 g / ml的区域中被回收，这通过感染力滴度，病毒基因组和融合糖蛋白峰的重合得到证实。对回收率的分析表明，使用碘克沙醇可将病毒产率提高至69％。还发现碘克沙醇对用于感染性测定的HeLa细胞无毒，从而无需通过透析将其下游去除。
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