Preparation of mouse monoclonal antibody

For generation of the mouse mAb against H7N9 rHA, two BALB/c male mice were immunized through intraperitoneal injection one time with a mixture of 50 μg purified H7N9 rHA (for generation of 1C6B) or rHA1 (for generation of F3-2) and complete Freund’s adjuvant (Sigma-Aldrich, Product Number F5881) and three booster injections in a 2-week interval with a mixture of 25 μg purified H7N9 rHA or rHA1 and incomplete Freund’s adjuvant (Sigma-Aldrich, Product Number F5506) followed by a final booster injection of 25 μg purified H7N9 rHA or rHA1 in phosphate buffered saline (PBS). The procedures for screening of hybridoma were performed as described previously (Chen et al. 2002). Briefly, Sp2/0-Ag14 cells (ATCC CRL-1581) were mixed with mouse splenocytes derived from the mouse immunized with the purified H7N9 rHA or rHA1 in a 1:5 ratio of cell numbers. The 0.7 mL of polyethylene glycol 1500 (Sigma-Aldrich, USA) was added to the cell mixture and incubated in a 37°C water bath for 2 min with gentle shaking. The additional 10 mL of Dulbecco’s Modified Eagle Medium (DMEM) was then added to the cell mixture within 4 min. After centrifugation, cells were collected and resuspended in 30 mL of DMEM containing 15% fetal bovine serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate (Thermo Scientific, USA), and HAT Media Supplement (Sigma-Aldrich, USA). Fusion cells (0.1 mL) were aliquoted into each wells of 96-well cell culture plates and grown at 37°C in the 5% CO₂ incubator. The 50 μL of HT Media Supplement (Sigma-Aldrich, USA) was added to each wells of 96-well cell culture plates on day 7 after cell fusion. The culture media were collected on day 14 for analysis of the target antibodies by ELISA using 100 ng of purified H7N9 rHA as the antigen. The hybridoma cells which can produce the antibodies against H7N9 rHA were further subcloned into a monoclonal antibody by limiting dilution method.