H3N2-specific VHH genes were selected from the sdAb library. (A) The enrichment for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. Phages collected from each round were incubated with H3N2 virus and NaHCO3 (100 mM, pH 8.0), respectively. Then, the eluted phages were transformed into TG1 cells. +: Phages transformed into cells after panning with H3N2 virus. −: Phages panning with NaHCO3 were used as control. (B) After three rounds of panning, H3N2-specific VHHs were enriched 67-fold compared with control (+/−). (C) Periplasmic extract ELISA for 95 colonies. The colonies, whose absorbance signal (nanobodies incubated with H3N2) is more than twofold higher than that for the negative control (nanobodies incubated with NaHCO3), were considered as positive. (D) Four kinds of different amino acid sequences of anti-H3N2 VHHs were identified.