Elisa coating solution

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.

Orbital blood sampling was used to collect serum from mice at 7, 14, and 21 days after immunization. Indirect ELISA was then used to detect IgG antibody levels in mouse sera. ELISA coating solution (Solarbio, China) was used to dilute the antigen to a working concentration; and FC (4.5 ng/ml), GS (2.6 ng/ml), and FCGS (3.0 ng/ml) were added to 96-well plates and kept overnight at 4°C. Each well was washed three times with PBST, 5% skim milk was added, and the plates incubated at 37°C for 2 h. The liquid in each well was then discarded, wells were washed with PBST, and mouse serum (1:2,000) was added and again incubated at 37°C for 1 h. After being washed with PBST, rabbit anti-mouse IgG H&L (HRP) (1:5,000) (Abcam, Cambridge, UK) was added to each well and incubated at 37°C for 1 h. After being washed with PBST, a one-component TMB substrate color developing solution (Solarbio, China) was added, and plates were maintained at room temperature in the dark for 15 min. An ELISA stop solution (Solarbio, China) was then added to each well, and the absorbance at 450 nm (OD value) was measured with a microplate reader within 5 min.