Adenovirus Titration and Quantification

For infectivity assays, virus for titration was serially diluted in DMEM supplemented with 10% FCS and 50 μl of inoculum was added per well of a 96 well plate containing sub-confluent HEK293A cells. After 24 hours a further 50 μl media was added to cells, and after 48 hours infectivity was assessed. For GFP expressing viruses, the GFP positive cells were visualised directly and counted by fluorescent microscopy. For marker-less viruses, an anti-hexon immunostaining assay based on the QuickTiter™ Adenovirus Titer Immunoassay kit (Cell Biolabs Inc) was used [22] (link). Cells were fixed at −20°C in methanol, before the plates were blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Adenovirus hexon protein was stained by addition of 50 ul per well primary mouse anti-hexon polyclonal antibody (Cell Biolabs Inc.) followed by 50 ul secondary anti mouse horseradish peroxidise (HRP) conjugated antibody. Staining was developed by addition of 3,3′ diaminobenzidine (DAB) substrate, and positive brown stained cells in the wells were counted by light microscopy. For detection of Matrix Protein 1 (M1) antigen an anti-M1 monoclonal antibody (Abcam, Cambridge, UK) was used. Viral particles were measured by spectrophotometry as described previously [23] (link).