Anc80L65 Neutralization Assay in CSF and Serum

Antibody titers against Anc80L65 in CSF and serum were determined through neutralization assays^{3 (link)}. Using a 96-well format, heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free medium (Life Technologies, Carlsbad, CA), and then treated with Anc80L65-luciferase (10⁶ GC/well) for 1 hour at 37 °C. The sample/Anc80L65-luciferase mix was then transferred onto HEK293 cells, which were treated with adenovirus (MOI 20) the day before. After 1 hour at 37 °C, diluted serum medium (1 part serum-free, 2 parts with serum) was added to each well. Two days later, the cells were treated with lysis buffer (Promega, Madison, WI) and frozen at −80 °C for 30 minutes. The cells were then thawed at 37 °C for 15 minutes before being treated with substrate buffer (Tris-HCl, MgCl₂, ATP [Life Technologies, Carlsbad, CA], D-Luciferin [Caliper Life Sciences, Hopkinton, MA]). Luminescence output was read using the Synergy BioTek Plate Reader (BioTek, Winooski, VT).