Sprangers, M.C., Lakhai, W., Koudstaal, W., Verhoeven, M., Koel, B.F., Vogels, R., Goudsmit, J., Havenga, M.J. and Kostense, S.
Notes: To help determine the usefulness of a recombinant Adenovirus for gene delivery and vaccination, the authors compared various methods to determine the amount of anti-Adenovirus serotype 5 (Ad5) neutralizing activity present in human sera. This study describes a system using firefly luciferase in Ad5 vector to infect A549 human lung carcinoma cells. Using a range of 8,000 to 5 virus particles/cell, the Ad5-luciferase was added to 104 cells/well in a 96-well plate in a total volume of 200µl. After 24 hours, the medium was discarded, and 100µl of PBS was added to each well followed by 100µl Steady-Glo® Luciferase Assay Reagent. After 15 minutes incubation at room temperature, 100µl from each well was transferred to a black and white isoplate and luminescence was measured on a 1450 Microbeta Trilux. An alternate neutralization assay method used the MTT-based CellTiter 96® Non-Radioactive Cell Proliferation Assay to score the Ad-mediated cytopathic effect by staining for viable cells and subsequent analysis of optical density. (3095)