Goat anti chicken horseradish peroxidase antibody

Sucrose gradient-purified viruses were diluted in PBS and added to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 hemagglutinating units (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 in chicken were then added in serial twofold dilutions with PBS containing 0.05% Tween-20, 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) [22 (link)].

The ELISA was performed as previously described [29 (link)], and 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 overnight incubation at 4 °C, the samples in wells were blocked with PBS-nonfat dry milk, and the antisera against the F/98 virus, the rF/HA_S127N virus or the rF/HA_A180V virus in chickens were then added in serial twofold dilutions with PBS containing 0.05% Tween 20 and incubated for 3 h at 37 °C.
After washing, goat anti-chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and incubated for 1.5 h at 37 °C, TMB (3,3′,5,5′ tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H₂SO₄. The absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT, USA), and the area under the curve (AUC) of either virus was assessed for virus-Ab binding above that of the corresponding negative control with GraphPad Prism 8 software (San Diego, CA, USA).