Fig. 2: Identification of two neutralizing nanobodies (Nb14-Fc and Nb32-Fc) targeting two different epitopes on HSV gD.

a, b Multi-concentration ELISA-binding assay of Nb14-Fc (a) and Nb32-Fc (b) towards HSV gD. The OD₄₅₀ emissions are depicted by curves. EC₅₀ (nM) indicated median effect concentration and was calculated to assess the binding potency of Nbs. c, d The binding kinetics of Nb14-Fc (c) and Nb32-Fc (d) to HSV-1 gD were monitored by the Biacore 8 K system. e, f The binding kinetics of Nb14-Fc (e) and Nb32-Fc (f) to HSV-2 gD were monitored by the Biacore 8 K system. The actual responses (colored lines) and the data fitted to a 1:1 binding model (black dotted lines) are shown in (c–f). K_D, equilibrium dissociation constant; k_a, association constant; k_d, dissociation constant. g, h The neutralizing activities of Nb14-Fc (g) and Nb32-Fc (h) against HSV-1 and HSV-2. The half-maximal inhibitory concentration (IC₅₀) values of the plaque reduction neutralization test (PRNT) were calculated by fitting the inhibition rates against antibody concentrations with a sigmoidal dose-response curve. i Competitive binding of Nb14-Fc and Nb32 to HSV-2 gD detected by ELISA. HSV-2 gD was coated on 96-well plates, Nb14-Fc was mixed with 4-fold serial dilutions of Nb32. The competition was determined by the reduction of the HRP-anti-IgG1 Fc induced chemiluminescence signal (OD₄₅₀). The inhibition was calculated by comparing it to the Nb negative-control well. Data and error bars are mean ± S.D, n = 3 biological independent experiments in (a, b, and g–i). Nb, Nanobody. Source data are provided as a Source Data file.