Rapid Fluorescent Focus Inhibition Test (RFFIT)

Immunogenicity determination of rabies vaccines or natural infection-elicited antibody responses against rabies virus is determined using serological assays including the rapid fluorescent focus inhibition test (RFFIT). This method is also used to verify the effect of compounds. A 100-μL serial dilution of each test compound (9 dilutions in a 3-fold stepwise manner) was incubated with 50 μL of RABV CVS (20, 000 FFU/well) in duplicate for 1 h at 37°C. After neutralization, 50 μL of BSR cells (1 × 10⁶/ml) were added into each well, then plates were cultured for 24 h in a 5% CO₂ incubator at 37°C. Finally, cells were fixed with pre-chilled 80% acetone at 4°C for 30 min and stained with FITC-conjugated anti-rabies N monoclonal antibody (Fujirebio Diagnostics, Malvern, PA) at 37°C for 30 min (Smith et al., 1973). The fluorescent intensity per well was recorded visually under a fluorescence microscope (Olympus, Tokyo, Japan).