The use of double nanobody sandwich method for influenza A grade 2 detection. (A) We used the magnetic beads linked with streptavidin to capture Nb3-biotin and used the Nb1 coupled to HRP as detector for H3N2 antigen detection. (B) Serial concentrations of H3N2 antigen were used for their detection. The lowest antigen concentration that could be detected was 50 ng/mL (P < 0.05). (C) The relative activity of Nb1 and Nb3 which were incubated at 37°C for different amounts of time. The activity of Nb1 and Nb3 reduced 26.03% and 17.22%, respectively, after heat incubation for 48 h.