Purification of H3N2-specific nanobodies and specificity of two paired nanobodies. (A) Four nanobodies having different sequences were purified by immobilized metal affinity chromatography (IMAC) using a His-Select column. (B) Isoelectric point, molecular weight, and yield of four nanobodies. (C, D) The Nb1 and Nb3, recognizing two different epitopes as shown via a pairing experiment, were chosen to detect the specificity to five types of different viral proteins by ELISA. One hundred microliters of each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates, and 100 μL nanobodies (10 μg/mL) were added. After reaction with mouse anti-HA tag antibody and then anti-mouse IgG-alkaline phosphatase, the chromogenic solution containing bisphosphate was added, and the absorbance at 405 nm was measured by an ELISA reader. BSA (5 μg/mL) was used as control. Values were the means of three replicates. The values of Nb1 and Nb3 were significantly different from the control (***P < 0.001). (E) Specificity of the nanobodies to the proteins on the surface of the H3N2 virus by ELISA. The H3N2 virus and HA protein were coated, and BSA was used as control. After incubation with Nb1 and Nb3, respectively, anti-His tag mouse monoclonal antibody was added, and then anti-mouse IgG-alkaline phosphatase was added for detection. Finally, absorbance at 405 nm was read.