2.3 Antisera and monoclonal antibody preparation.
Antisera from ferrets against GX/18 or GD/104 were obtained from our previous study(Yang et al., 2016), and counterparts from guinea pigs were generated by using six-week-old animals (Vital River Laboratories, Beijing, China) that were serologically negative for circulating seasonal influenza A and B viruses. Two guinea pigs were intranasally inoculated with 106EID50 of each virus in a volume of 300 μl (150 μl per nostril), and the antisera were collected 3 weeks after inoculation by euthanizing the animals. After being treated with vibrio choleraereceptor-destroying enzyme (DenkaSeiken) for 18h at 37°C and heat-inactivated at 56°C for 30 min, the antisera were ready for HI assay. The mAb used in this study was produced according to the standard protocol previously described by Li et al .(Li et al., 2017). Briefly, BALB/c mice were immunized intraperitoneally with 500µl β-Propiolactone-inactive GX/18 virus (with 28hemagglutination unit) at 6-, 9-, and 11-weeks old. Three days after the final immunization, the mice were euthanized and splenocytes were collected. The spleen cells were fused with Sp2/0 cells to produce a hybridoma as described by Gefter et al .(Gefter et al., 1977), and cloned in 96-well plate with HAT medium. The positive hybridoma clones were screened through HI assay and then subcloned four times by limiting dilution method. Finally, the cloned hybridoma cells were injected into the peritoneum of the BALB/c mice, and abdominal ascites were collected 7 days later.