Tandem competition assays were used for binning mAbs to CCHFV GP38 recombinant protein. Nickel-charged tris-nitrilotriacetic acid (Pall ForteBio part number 18-5101) sensors were loaded with rGP38his recombinant protein, equilibrated for 10 min in water, then 10 mM nickel chloride for 60 s, and washed for 60 s in PBS. Sensors were then loaded with rGP38his recombinant protein (10 μg/ml) by 5-min incubation in 1× kinetics buffer (Pall ForteBio). Baseline readings were determined by equilibrating sensors for 60 s in 1× kinetics buffer. Purified mAbs against GP38 were diluted with 1× kinetics buffer to a concentration of 100 nM. Two null antibodies (EBOV-H3C8 and CCHFV-11E7) were also diluted and tested. One column of the Octet sample plate was used for seven mAbs, and each assay included a no-antibody control well. The eight sensors were incubated in the saturating antibody wells for 5 min before moving the sensors to a baseline well for 60 s in 1× kinetics buffer. The sensors were regenerated by incubating them in solution of 10 mM glycine with a pH of 2.0 for 10 s, followed by 10 s in PBS (pH 7.4). This regeneration cycle was performed three times before moving the sensors to a 1-min PBS wash. After washing, sensors were recharged with a 1-min incubation in a 10 mM nickel chloride solution. The sensors were then stored in water before using in additional assays. The data from the sensors were analyzed using the binning function of the Octet analysis software and competition groups assigned.

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