Goat anti chicken iga h l hrp

On the 28th dpv, 8-cm-long fresh tracheas and 10-cm samples of the small intestines (including the Peyers’ patches) were repeatedly washed with 500 μL cold PBS containing protease inhibitor to obtain lavage fluid, and were then centrifuged at 10,000× g for 20 min at 4 °C to remove precipitated impurities before being stored at −20 °C. The concentrations of total sIgA in the lavage fluid were individually monitored using the direct sandwich ELISA kit (SinoBestBio, Shanghai, China) according to the manufacturer’s instructions. For detection of the titer of IBV-specific sIgA in the lavage fluid, IBV-coated 96-well ELISA plates were used to bind the IBV-specific sIgA with 100-μL lavage for each well and three-well repeats for each sample, which were then incubated at 37 °C for 1 h. After washing five times, 100-μL goat anti-chicken IgA H&L (HRP) (Abcam Ltd., Shanghai, China) at a 1:2000 dilution was added to each well and incubated at 37 °C for 1 h, followed by washing. Finally, 10 μL TMB (3,3′,5,5′-Tetramethylbenzidine dihydrochloride hydrate) Chromogen Solution (Beyotime Biotechnology Ltd., Shanghai, China) was added to each well, which were incubated at room temperature for 10 min without light, and then the plates were read at the wavelength of 630 nm.