Fig. 9: P17-tagged G12-scFv showed stronger antigen binding affinity and specificity and HBV-neutralizing activity than G12-scFv.
a Biolayer interferometry to determine the binding affinity of G12-scFv-HA and G12-scFv-HA-P17 proteins to recombinant HBV small surface protein from CHO cells. Kd, equilibrium dissociation constant. b Enzyme-linked immunosorbent assay (ELISA) to test binding affinity and specificity of G12-scFv-HA and G12-scFv-HA-P17 proteins. The pooled sera of chronic hepatitis B patients and healthy donors were confirmed to be positive and negative for hepatitis B surface antigen (HBsAg) by a commercial KHB ELISA kit, and then reacted with the microtiter wells coated with 5 μg/ml solutions of G12-scFv-HA and G12-scFv-HA-P17, respectively. Data are presented as mean values ± SD. The statistical significance of differences between two experimental groups was assessed by one-way ANOVA. c Determination of HBV-neutralizing activity of G12-scFv-HA and G12-scFv-HA-P17 proteins by northern blotting and ELISA. HepG2-NTCP cells were inoculated overnight with 100 vge/cell HBV particles pre-incubated for 30 min with serially diluted G12-scFv-HA or G12-scFv-HA-P17 protein. After extensive washing, cells were cultured in DMEM containing 2.5% DMSO. Intracellular viral RNAs (left panel) and secreted HBeAg (right panel) in supernatants were detected at day 5 post-inoculation by northern blotting and ELISA, respectively. Pc RNA, precore mRNA; pgRNA, pregenomic RNA. For comparison, the signal intensity of pc/pgRNA was normalized to that of 28 s and 18 s rRNA (as loading controls). A450 nm values for HBeAg subtracting the ELISA cutoff of 0.11 at various concentrations of scFvs, determined by four independent experiments, are shown. IC50 was calculated with Origin 8.0 software. Pc/pgRNA and HBeAg from untreated cells were set at 100%. +, with HBV inoculation; −, without HBV inoculation.
