Fig. 3

Functional validation of the screened Nbs in vitro. a Molecular docking models of Nbs with FGFR4. The structure of light blue represents antigen, the structure of dark blue represents Nbs, the interface regions of red and green represent the region of the Nb in contact with antigen, ΔG denotes the free energy of binding. b Schematic diagram of expression vector encoding Nb-Fc. c Detection of the binding ability of Nbs by antibody-antigen binding ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by the Student’s t-test. d Detection of the binding ability of Nbs by antibody gradient dilution ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by two-way ANOVA. e Detection of the binding affinity of Nbs by SPR assay. f Evaluation the specificity of Nbs by antibody-antigen binding ELISA. Data were analyzed by one-way ANOVA. The experiments were performed independently in triplicate. Data are expressed as mean ± SEM. ****p < 0.0001