Elisa plates

The four purified anti-S mAbs were examined to select the optimal one for viral antigen capture. In detail, each purified anti-S mAb (mAb9, mAb10, mAb17, or mAb18) diluted in Solarbio ELISA coating solution (8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/mL; 100 μL/well) was coated on 96-well ELISA plates (NEST, Wuxi, China) at 4 °C overnight. Then, the plates were washed five times with PBST and blocked with 5% skim milk at 37 °C for 2 h. After washed, each coated well was incubated with 100 μL inactivated cell-cultivated PEDV supernatants (1 mg/mL) at 37 °C for 1 h to capture viral antigens. The plates were washed and incubated with the generated rabbit anti-PEDV pAbs at 37 °C for 1 h. Following washed, each well was incubated with 100 μL 1:1000 diluted HRP-conjugated goat anti-rabbit IgG (Abcam, Cambridge, England) as secondary antibody at 37 °C for 30 min. After washed, each well was reacted with 100 μL TMB substrate solution (Solarbio) in dark at RT for 5 min. After added with 100 μL/well of ELISA stop solution (Solarbio), OD₄₅₀ values were immediately measured and recorded on a microplate reader (BMG-Labtech, Offenburg, Germany). The anti-S mAb with the highest OD₄₅₀ values was chosen to establish ELISA. Three replicates of each sample were run, and each experiment was independently repeated three times.

Blood was obtained from the retro-orbital plexus using a capillary tube, and collected in an Eppendorf tube. After centrifugation (5000× g for 15 min), the sera was stored at −20 °C. Then, 40 ng purified gE (Abcam) was adsorbed onto ELISA plates (NEST, Wuxi, China) overnight in PBS at 4 °C and blocked with 10% FBS in PBS at 37 °C for 2 h. Then, the serially diluted sera were added to the plates and incubated for 1 h at 37 °C. After the plates were thoroughly washed using PBST, HRP-conjugated anti-mouse IgG was further added into the plates (1:5000, Biodragon Immunotechnologies, Beijing, China), and allowed to incubate for another 1 h at 37 °C. Finally, 100 μL of TMB soluble (Biodragon Immunotechnologies) was used for the color reaction, and the reaction was terminated via the addition of 50 μL 2 M H₂SO₄. The absorbance was measured at 450 nm using a BioTek plate reader (BioTek, Winooski, VT, USA).