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.

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