FIG 2.

Characterization of MERS-CoV RBD-specific NbMS10 and NbMS10-Fc Nbs. (A) SDS-PAGE and Western blot analyses of purified NbMS10 and NbMS10-Fc. The Nbs were subjected to SDS-PAGE (left) or Western blotting (right), followed by detection using anti-llama antibody. The molecular weight marker (in kDa) is indicated on the left. (B) Detection of binding between NbMS10 or NbMS10-Fc and MERS-CoV S1 (MERS-S1) or RBD (MERS-RBD) protein by ELISA. The plates were coated with MERS-CoV S1-His or RBD-Fd protein (2 μg/ml), followed by sequential incubation with respective Nbs and goat anti-llama and HRP-conjugated anti-goat IgG antibodies. The data are presented as mean A₄₅₀ values ± the standard deviation (SDs) (n = 2). Significant differences (*; **, and ***) are shown in the binding of Nbs to MERS-S1 or MERS-RBD at various concentrations. (C) The binding kinetics between NbMS10 or NbMS10-Fc and MERS-CoV RBD or S1 protein were measured by SPR. MERS-CoV RBD-Fc protein was used for binding to NbMS10 (containing a C-terminal His₆), and S1-His protein was used for binding to NbMS10-Fc (containing a C-terminal human Fc). (D) Detection of NbMS10 and NbMS10-Fc neutralizing activity against MERS-CoV infection (EMC2012 strain) by a microneutralization assay. The Nb-MERS-CoV mixtures were incubated with Vero E6 cells and observed for the presence or absence of CPE. Neutralizing activity of Nbs was recorded as the concentration of Nbs in complete inhibition of MERS-CoV-induced CPE in at least 50% of the wells (ND₅₀). The data are expressed as mean ND₅₀ ± the SD (n = 3). The experiments were repeated twice, and similar results were obtained. The “(−) control” in panels A, B, and D refers to SARS-CoV 33G4 mouse MAb.