Figure 2
From: In situtransduction of target cells on solid surfaces by immobilized viral vectors
Immobilization efficiency of biotinylated adenoviral vectors on streptavidin-coated wells. (A) Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (1 × 106 – 5 × 106 viral particles in 50 μl PBST per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. The solution of each well, which contained unbound viral particles, was collected and analyzed for the infectivity on D-17 cells (○). The same numbers of biotinylated Ad5.CMV-LacZ without application to streptavidin-coated wells were also analyzed for the infectivity on D-17 cells (●). The infectivity of immobilized viral particles was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied to each well. The percentage of immobilized viral particles in the total is shown at the top. Each data point shown is the average + SD (n = 3). (B) Biotinylated Ad5.CMV-LacZ, prepared as above, was incubated in streptavidin-coated wells (2.5 × 106 viral particles per well) for 30 min at 25°C. The solution of each well was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was incubated in the same manner as above. These steps were repeated several more times. The solution in the well, which contained unbound viral particles, was collected after each addition cycle and titrated on D-17 cells (open bars). The Control bar shows the total infectivity of biotinylated Ad5.CMV-LacZ that was applied to wells at each addition cycle. The infectivity of immobilized viral particles (solid bars) was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied at each addition cycle. The percentage of immobilized viral particles in the total at each addition cycle is shown at the top. Each data point shown is the average ± SD (n = 3).