Preparation of PBS emulsion (200 ~ 400μl/ mouse) containing 25 ~ 100 μ g antigen and Freund's complete adjuvant. Connect two syringes with a connector with a double-headed plug. One syringe contains antigen and the other contains adjuvant. Push the syringe back and forth for 5 ~ 10 min until a stable emulsion is obtained. Mice (3 ~ 5 mice per antigen) were injected intraperitoneally with a 22g needle, and then an emulsion (200 ~ 400μ l) was prepared with equal volume PBS containing 10 ~ 50μ g antigen and Freund's incomplete adjuvant. Seven days after the second immunization, the tail of mice was cut off by 0.5 cm with sterile scissors or shaving blades, and blood was collected. Collect 100 ~ 200μ l blood in 1.5 ml microcentrifuge tube. It is convenient to collect blood by keeping mice warm for 30 ~ 60 s under a heating lamp or squeezing their tails with their thumb and forefinger. After the blood clot is formed, remove the blood clot from the test tube and surface with a wooden smear, and do not break the blood clot. After removing the blood clot, put the serum into another microcentrifuge tube with a 200μl pipette. Serum antibody titer was determined by ELISA. If necessary, the antibody was further confirmed by protein blot. If the titer is too low to be used for fusion (≥11000), the mice will be immunized every two weeks until sufficient response is obtained. If blood is collected more than 3 times, cut a tail vein with a shaving knife to collect blood. When the antibody titer is high enough (≥11000),10 was injected intraperitoneally (200 ~ 400μ l) or subcutaneously via tail vein (50 ~ 100μ g) 3 days before fusion, but 2 weeks after previous immunization.
Cell fusion was performed after 3 days of intensive immunization.