Quantification of AAV Particle Titers by Infrared Fluorescence Scanning of Coomassie-Stained Sodium Dodecyl Sulfate-Polyacrylamide Gels
Quantification of AAV Particle Titers by Infrared Fluorescence Scanning of Coomassie-Stained Sodium Dodecyl Sulfate-Polyacrylamide Gels
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DOI：
10.1089/hgtb.2012.049
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发表时间：
2012-06-01
影响因子：
--
通讯作者：
Weber, Thomas
中科院分区：
文献类型：
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作者：
Kohlbrenner, Erik;Henckaerts, Els;Weber, Thomas
基于腺相关病毒（AAV）的载体作为基础和临床前研究以及人类基因治疗试验中的基因递送载体已经获得越来越多的关注。特别是对于后两种-出于安全性和治疗功效的原因-载体制剂的所有相关参数的详细表征是必不可少的。常规用于分析重组AAV载体的两个重要参数是（1）含有（重组）病毒基因组的病毒颗粒的滴度和（2）载体制备物的纯度，最常见的是通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）随后进行银染色来评估。一个重要的第三个参数，总病毒颗粒的滴度，即含基因组和空病毒衣壳的组合滴度，很少被确定。在这里，我们描述了一种简单而廉价的方法，允许同时评估载体纯度和总病毒颗粒滴度的测定。通过与已建立的病毒颗粒滴度测定方法进行比较，对该方法进行了验证，该方法基于考马斯亮蓝与蛋白质结合时在红外光谱中发出荧光的事实。通过SDS-PAGE分离病毒样品，然后进行考马斯亮蓝染色，并用红外激光扫描装置进行凝胶分析。结合蛋白质标准品，我们的方法可以快速准确地测定病毒颗粒滴度，同时评估载体纯度。
Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.