Goat anti chicken iga

The levels of specific IgG in sera and sIgA in jejunal lavage fluid were detected by indirect ELISA as previously described (24 (link)). Briefly, ΔHexon protein (1 mg/mL) was coated on the 96-well plate (100 μL per well). The prepared sera diluted at 1:50 and jejunal lavage fluid diluted at 1:10 were added, respectively, and incubated at 37°C for 1 h. HRP-conjugated goat anti-chicken IgG or goat anti-chicken IgA (Abcam, USA) were used as secondary antibodies, respectively. 100 μL of o-phenylenediamine (1 mg/mL) and 0.01% H₂O₂ were added to each well, and the reaction was stopped by 2 M H₂SO₄. The absorbance was measured at 490 nm using a reader (Bio-Rad, USA). Each sample was tested in triplicate.

Purified the H5N8 HA proteins were coated onto 96-well Maxisorp clear plates at 1 mg/mL in 50 mM Na₂CO₃, pH 9.6, overnight at 4°C. 500μL serum or PBS buffer-diluted anal swabs were incubated at 37°C for 40 minutes. Plates were washed 3 times with PBST (0.5% Tween-20), and then incubated with HRP-goat anti-chicken IgG (Solarbio, Beijing, China) or goat anti-chicken IgA (Abcam, USA). After 40 minutes incubation at 37°C, plates were washed 3 times with PBST (0.5% Tween-20) and 1-Step Ultra TMB-ELISA (Solarbio, Beijing, China) was added. Following 12 minutes incubation, reactions were stopped with 2 M sulfuric acid. The absorbance of each well at 405 nm was determined using ELISA Reader (Biored, USA).