High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype.
Accurate adenovirus (Ad) quantification requires labor- and time-intensive viral stock purification. While crude viral lysates can be titered by plaque assay, this cell-based assay is neither rapid nor accurate. Consequently, a method for quantification of crude, unpurified viral culture lysates is needed. Given growing interest in alternative Ad serotypes (different from well-studied and characterized serotype Ad5) for basic research and for therapeutic applications, such a method should also apply to alternative serotypes. Using a Q Sepharose XL (QSXL) column-based method, we describe a robust quantification method resulting in efficient retention of viral particles of all serotypes, while non-viral components of crude infected cultures remain largely in the flow-through. The high-performance liquid chromatography-QSXL method allows rapid, accurate adenoviral quantification in crude lysates as well as identification of the various serotypes present in mixed-serotype crude lysates. We also report on conditions that efficiently strip and regenerate the column, extending its functional life.
Kuhn
I
I
Berlex Laboratories, Gene Therapy Department, Richmond, CA 94806, USA. Irene_Kuhn@Berlex.com
Larsen
B
B
Gross
C
C
Hermiston
T
T
D000256:Adenoviridae / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D012703:Serotyping / Q000379:methods; D014771:Virion