Screening sdAbs Binding to DIII Regions

Purified sdAbs (1 μl) were spotted on NC membranes, and non-specific binding sites were blocked with 5% BSA dissolved in tris-buffered saline (TBS; pH = 7.3, Sigma-Aldrich). After a wash with TBS containing 0.05% Tween 20 (TBST-20; 5 min), membranes were separately incubated (1 h) with either biotinylated DIII-1^G299–K307 or DIII-2^V371–R388 reconstituted in a phosphate buffer (1 μg/ml, pH = 7.0). After three washings, streptavidin-HRP conjugate (1:30,000 in TBST-20; Sigma-Aldrich) was added, and the membranes were incubated for 1 h. Subsequently, five washes with TBST-20 (5 min each) and one with TBS (5 min) were performed before incubating the membranes with a SuperSignal West Dura chemiluminescent substrate, and the signal was recorded on a C-DiGit Blot Scanner (Odyssey CLx, Cambridge, United Kingdom). Simultaneously, sdAbs raised against N. meningitides [VHH_F3 (Kulkarni et al., 2020 (link)): non-related sdAbs; negative control] and hyperimmune serum of horse surviving natural WNV infection (positive control) spotted on NC membranes, were included in the assay to undergo incubations with DIII-1^G299–K307 or DIII-2^V371–R388, streptavidin-HRP conjugate, and chemiluminescent substrate, followed by signal detection.