Hemagglutinin‑inhibition (HI) assay and
Microneutralization (MN) assay
Antisera were treated with cholera filtrate (Sigma-Aldrich, St. Louis,
MO, USA) to remove nonspecific hemagglutination inhibitors before HI
assay. HI assay was performed using 4 hemagglutination units (HAU) of
H9N2 and 1% (v /v) chicken erythrocytes as we described
previously (Peng et al., 2019).
MN assay was performed as previously described (H. Zhu et al., 2018).
Briefly, the sera were serially diluted with 100 TCID50virus and incubated at 37 °C with 5% CO2 for 1 h. The
serum-virus mixtures were added to MDCK cells and incubated for 1 h.
After incubation, the serum-virus mixtures were removed. Serum-free DMEM
containing 2 µg/mL TPCK‑ trypsin was added to each cell and incubated at
37°C and 5% CO2. After 72 h of incubation, culture
supernatant was mixed with equal volume of 1% (v /v) chicken
erythrocytes to confirm the existence of hemagglutination by virus. The
MN titer was defined as the highest dilution of serum with absence of
hemagglutination.