*2.3. Immunization and Cell Fusion*

All animal experiments were implemented in accordance with the animal ethics committee (HZAUMO-2021-0184) and following principles authorized by the Huazhong Agricultural University animal experiment center. Immunogens (A1, A2, A3) and two different antigen doses (50 μg and 100 μg) were inoculated into Female Balb/c mice, 6 weeks old. For the first immunization, immunogens were mixed with FCA and given subcutaneous multipoint injection in the back or neck of mice. Subsequently, the immunogens emulsified with FIA were used to enhance immunization every 14 days. The serum titer and sensitivity were measured by ic-ELISA. A mouse with high serum titer and sensitivity was selected and immunized with immunogens without Freund's adjuvant for booster immunization. The mouse was euthanized and spleen cells were prepared for cell fusion. Activated SP2/0 myeloma cells and immunized spleen cells were fused at a ratio of 1:5~10 in 0.8 mL of 50% polyethylene glycol. The fusion cells were gently mixed in 1% HAT medium which contain feeder cells, then injected into 96-hole cell culture plates.
