Enzyme‑linked immunosorbent assay (ELISA)
ELISA assay was performed as we described previously (Peng et al.,
2019). Sucrose gradient-purified viruses were diluted in PBS and added
to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 HAU per
well. After incubation overnight at 4°C, samples in wells were blocked
with PBS-nonfat dry milk. Antisera against the F/98 virus, the
rF/HAS145N virus or the rF/HAA198V virus
in chickens were then added in serial twofold dilutions with PBS
containing 0.05% Tween 20, respectively, and incubated for 3 h at 37°C.
After washing, goat anti chicken horseradish peroxidase antibody (Abcam,
Cambridge, MA) was added and allowed to incubate for 1.5 h at 37 °C.
After washing, TMB (3,3′,5,5′ Tetramethylbenzidine) (Sigma, St. Louis,
MO, USA) substrate was added, and the reaction was stopped by adding
H2SO4. Absorbance was recorded at 450 nm
using an automated ELISA plate reader (model EL311SX; Biotek, Winooski,
VT). The area under curve (AUC) of either virus was assessed for
virus-Abs binding with GraphPad Prism 8 software (San Diego, CA) above
that of the corresponding negative control.