Cole Kenneth D, Pease Leonard F, Tsai De-Hao, Singh Tania, Lute Scott, Brorson Kurt A, Wang Lili
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
J Chromatogr A. 2009 Jul 24;1216(30):5715-22. doi: 10.1016/j.chroma.2009.05.083. Epub 2009 Jun 6.
Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and phiX174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed.
开发和校准检测设备及仪器需要病毒参考材料。我们使用电喷雾差分迁移率分析（ES-DMA）和定量氨基酸分析（AAA）来测定三种小型模型病毒（噬菌体MS2、PP7和phiX174）的颗粒浓度。使用噬菌斑测定、变性凝胶电泳和尺寸排阻色谱法测量病毒样品的生物活性、纯度和聚集情况。ES-DMA通过使用金纳米颗粒作为内标来对病毒颗粒进行计数。ES-DMA还能对病毒样品的大小和聚集程度进行定量测量。定量AAA也用于测定纯病毒样品中病毒蛋白的质量。将样品水解后，利用回收良好的氨基酸质量来计算样品中病毒颗粒的等效浓度。对于这些纯化样品，通过ES-DMA测定的病毒样品浓度与AAA预测的浓度高度一致。本文讨论了ES-DMA和AAA在表征病毒参考材料方面的优缺点。
