Aav titration elisa

Capsid titers were determined using the AAV Titration ELISA (Progen) according to the manufacturer's instructions. Genomic titers of rAAV-2 particles were determined using quantitative realtime PCR (qPCR) as described previously54 (link). Briefly, an aliquot of purified viral vectors was supplemented with 5 U DNase I and DNase I buffer (Fermentas), incubated for 3 h at 37°C, heat inactivated for 10 min at 65°C, and finally diluted 100-fold. 5 μl of these DNase I resistant particles containing the viral DNA supplemented with 7.5 μl Quantifast SybrGreen MasterMix (Qiagen) and CMV_forward/reverse primers55 (link) (10 μM each) were used as template in a qPCR reaction on a RotorGene cycler (Qiagen). Viral genome titers were calculated from a standard curve of 13–13 × 10⁶ copies of a CMV promoter-containing plasmid. Infectious titers were determined as described by Rohr et. al.55 (link), but using the Quantifast SybrGreen qPCR Kit (Qiagen) on a RotorGene qPCR cycler. Briefly, 10⁴ cells per well in a 24 well cell culture plate were seeded and incubated for 24 h with equal genomic titers of the viral particles. Subsequently, the cells were harvested, digested with proteinase K (Sigma) and residual single-stranded DNA was removed using S1 nuclease (Fermentas). Double-stranded DNA which originated from infectious particles harboring viral ssDNA56 (link) was then quantified using qPCR.