Viruses and cells
Anti-CCHFV and isotype antibodies
Mice
Passive protection experiments
Histology
In situ hybridization
Confocal microscopy
Microscopy
Cloning
GP38 purification
Flow cytometry
GP38 capture ELISA
DNA vaccination
Western blot
VLP production
Immunogold staining and electron microscopy of VLPs
Antibody competition
Statistical analysis
GP38his was diluted in 0.1 M carbonate buffer (pH 9.6) and plated in duplicate in the wells of a high-binding 96-well plate (Corning). Plates were blocked for 1 hour with PBST and 5% milk. Murine mAbs (ascites fluid) were serially diluted 10-fold (starting from 1:100) in PBST containing 5% milk. Murine mAb dilutions were incubated with GP38his 1 hour at 37°C. Plates were washed four times in PBST and incubated with an anti-mouse IgG conjugated to horseradish peroxidase (1:1000; Sigma-Aldrich) for 1 hour at 37°C. Plates were washed again four times in PBST, and 100 μl of 2,2′-azinobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) substrate (KPL) was added to each well. Reactions were stopped by adding 100 μl of ABTS stop solution of 5% (w/v) SDS. The OD at 405 nm was read on a Tecan microplate reader. End point titers were determined as the highest dilution with an absorbance value greater than the mean absorbance value from negative control antibodies (mouse ELISA, mAb-11E7, and anti-Junin virus GP1 mAb-QC03; or human ELISA and convalescent sera from an Ebola survivor) plus 3 SDs. Mean titers were plotted using GraphPad Prism 7 software.
For analysis of human sera, high-binding 96-well plates were coated with 500 ng per well of recombinant GP38, recombinant GN (Native Antigen), or recombinant N protein (Native Antigen) O/N, as described. The following morning, plates were blocked with Neptune blocking buffer (ImmunoChemistry Technologies) for 2 hours at 37°C. Plates were then washed and probed with half-log dilutions (starting at 1:50) of sera from convalescent human survivors of CCHF in Neptune blocking buffer for 1 hour at 37°C. After washing, anti-human IgG conjugated to horseradish peroxidase (1:1000; Sigma-Aldrich) was added for 1 hour at 37°C. Following a final wash, plates were treated with TMB substrate (SeraCare) at room temperature, and reactions were arrested using TMB stop solution. The OD at 450 nm was read on a Tecan ELISA plate reader, and mean titers were determined as stated above.