Automated elisa reader

Specific IgG in serum and IgA in mucosal suspensions were determined by enzyme-linked immunosorbent assay (ELISA) as previously described17 . In brief, ELISA plates were coated with the purified recombinant HA protein (our laboratory saved) in carbonate buffer (pH = 9.6) at 4 °C overnight. Plates were saturated with PBS containing 1% BSA at 37 °C for 2 h. The serum and mucosal suspensions of dilution degrees were added and incubated for 1 h at 37 °C. Subsequently, the HRP-conjugated goat anti-chicken IgG and goat anti-chicken IgA (BETHYL, CA) were added to the wells and incubated for an additional 1 h at 37 °C. Subsequently, a substrate solution containing o-phenylenediamine (OPD) and H₂O₂ was added, the reaction was allowed to proceed for 15 min at room temperature before it was terminated by the stop solution. Finally, the absorbance was measured at 450 nm, using an automated ELISA reader (Molecular Devices, Shanghai, CA).

Nonspecific SIgA and PEDV-specific IgG and SIgA were measured by ELISA according to the instructions as previously described [26 ]. Briefly, the ELISA plates were first coated with PEDV COE protein at 4 °C overnight and saturated with PBS (1% BSA) at 37 °C for 2 h. Second, the mucosal suspension and serum were added and incubated for another 1 h at 37 °C. Subsequently, HRP-conjugated goat anti-mouse IgG and goat anti-mouse IgA were added and incubated at 37 °C for 1 h. Third, a substrate solution containing H₂O₂ and o-phenylenediamine was added at room temperature for 15 min before termination. Finally, we used an automated ELISA reader (Molecular Devices, Shanghai, China) to detect the absorbance at OD₄₅₀ nm.