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.