Viruses and cells
Anti-CCHFV and isotype antibodies
Mice
Passive protection experiments
Histology
In situ hybridization
Confocal microscopy
Microscopy
Cloning
GP38 purification
Flow cytometry
GP38his ELISA
DNA vaccination
Western blot
VLP production
Immunogold staining and electron microscopy of VLPs
Antibody competition
Statistical analysis
mAb-13G8 or mAb-QC03 (2.5 μg/ml) were diluted in 0.1 M carbonate buffer (pH 9.6), plated on a high-binding 96-well plate (Corning, Corning, NY) and incubated overnight at 4°C. Plates were blocked for 2 hours in blocking buffer [PBS with 0.05% Tween 20 (PBST) containing 3% milk/3% goat sera] at 37°C. Plates were washed four times in PBST and incubated with supernatant from transfected 293T cells at the indicated dilution in blocking buffer for 2 hours at 37°C. Plates were washed four times in PBST and incubated with an anti–M-segment antisera from DNA-vaccinated rabbits (diluted 1:1200) in blocking buffer and incubated at 37°C for 1 hour. Plates were washed four times in PBST and incubated with anti-rabbit IgG conjugated to horseradish peroxidase (1:1000; KPL) for 1 hour at 37°C. Plates were washed again four times in PBST, and 100 μl of SureBlue Reserve TMB microwell peroxidase 1-component (KPL) was added to each well. Reactions were stopped by adding 100 μl of TMB stop solution (KPL). The optical density (OD) at 450 nm was read on a Tecan microplate reader (Tecan Group Ltd.).