Goat anti mouse iga biotin

ELISA analysis was carried out as previously reported (60 (link)): To determine eOD-specific or RBD-specific IgA and IgG titers, Costar Polystyrene High Binding 96-well plates (Corning) were coated with eOD antigen or RBD antigen at 2 μg/ml in PBS overnight at 4°C and blocked with blocking buffer (PBS + 2% BSA) for 2 hours at 25°C. Mouse serum samples were diluted in blocking buffer starting at 1:50 followed by 4× serial dilutions, and fecal samples were diluted in blocking buffer starting at 1:50 followed by 3× serial dilutions. Samples were incubated in plates for 2 hours at 25°C. To determine antigen-specific IgG antibodies, goat antimouse IgG–horseradish peroxidase (HRP; 1:5000; BioRad) was added to the wells and incubated for 1 hour. To determine antigen-specific IgA antibodies, goat antimouse IgA-biotin (1:5000; SouthernBiotech) was added and incubated for 1 hour followed by incubation with streptavidin-HRP (1:40,000; Thermo Fisher Scientific Pierce High Sensitivity Streptavidin-HRP) for 30 min. Plates were developed using a 3,3′,5,5′-tetramethylbenzidine substrate for 1 to 5 min and stopped using 2 M sulfuric acid, and the absorbance (450 nm with 540-nm reference) was measured on a plate reader. Endpoint cutoff titers are reported as inverse serum dilutions giving an HRP absorbance of 0.3 above background.