Hitt D C, Booth J L, Dandapani V, Pennington L R, Gimble J M, Metcalf J
Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Mol Biotechnol. 2000 Mar;14(3):197-203. doi: 10.1385/MB:14:3:197.
As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.
随着腺病毒载体在基因治疗方案中的应用不断增加，相应地需要快速、准确且可重复的滴度测定方法。目前存在多种用于测定重组腺病毒载体滴度的方法，包括光吸收法、电子显微镜法、荧光聚焦测定法以及“金标准”噬斑测定法。本文介绍了一种基于绿色荧光蛋白（GFP）表达的新型流式细胞术直接滴度测定方法，GFP是一种整合到多种腺病毒载体中的追踪标记物。将该方法与噬斑测定法进行比较，使用经氯化铯纯化的、表达GFP的腺病毒（AdEasy + GFP + GAL）进行10⁻⁴至10⁻⁶倍稀释。两种方法得到的滴度相似：分别为3.25 ± 1.85×10⁹ PFU/mL和3.46 ± 0.76×10⁹绿色荧光单位/（gfu/mL）。与噬斑测定法所需的7至10天相比，流式细胞术方法在24小时内即可完成。这些结果表明，GFU/mL是荧光标记腺病毒载体的一种替代功能滴度测定方法。
