PRNT and Inhibition Assays for Neutralizing Antibodies

We determined the titer of neutralizing antibodies by a standard plaque reduction neutralization titer (PRNT) assay using either BHK21 or SW13 cells^{23 (link)}. Results were plotted and the titers for 50% (PRNT₅₀) and 90% inhibition (PRNT₉₀) were calculated. The inhibition assay with J774.2 mouse macrophages was performed as follows: we mixed medium and E16 or E24 (2.5 μg of monoclonal antibody) with 5 × 10² PFU of WNV, incubated the mixture for 1 h at 4 °C, and then added to 5 × 10⁴ J774.2 mouse macrophages in individual wells of a 24-well plate. After 1 h, cells were washed four times with PBS to remove free virus and monoclonal antibody, DMEM with 10% FBS was added, and the cells were incubated for an additional 24 h. We subsequently harvested supernatants for a viral plaque assay on Vero cells.