Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included convent ional procedures initially developed to detect wild-type adenoviruses. Stan dard virological assays to quantitate adenoviruses rely on the virus to inf ect and replicate in the host cell until a cytopathic effect is observed. T he appearance of plaques, colonies of rounded, enlarged cells containing in fectious virions, usually takes 2 to 3 weeks to reach an endpoint. We descr ibe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determi ned. A fluorescent focus-forming assay was one of the first rapid adenovira l bioassays developed. Virus titer was determined using fluorescence immuno cytochemistry to detect adenovirus proteins and microscopy to count fluores cent foci in cultures of adenovirus-infected cells. In this study, we descr ibe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependen t appearance of hexon-positive cells. Adenovirus hexon detection in infecte d cells can provide data to determine virus titer, inducible promoter funct ion in vector-complementing cells, and vector replication in complementatio n-deficient cells, (C) 2000 Academic Press.