Sandwich ELISA for DAdV-3 Detection

To develop sandwich ELISA for detection of DAdV-3, mAb 3D9 was purified and HRP-conjugated as previously described (Wang et al. 2018 (link)). In the sandwich ELISA, the purified mAb 3D9 diluted in 0.1 M carbonate buffer with the final concentration of 1.25 µg/mL was coated into a 96-well ELISA plate (100 µL/well) at 4 °C overnight. The following morning, the 96-well ELISA plate was blocked with 360 µL of 5% skim milk in PBST for 2 h at 37 °C. After the blocking and washes, the virus supernatants or samples diluted were added into the 96-well ELISA plate for 1 h at 37 °C and the 96-well ELISA plate was washed with PBST for three times. Then, the HRP-conjugated mAb 3D9, diluted by PBST with a final concentration of 0.625 µg/mL, was added into the 96-well ELISA plate for 30 min at 37 °C. After washed with PBST for three times, the 96-well ELISA plate was added with 100µL TMB single-component substrate solution (Solarbio) per well and reacted for 15 min at 37 °C. Finally, 50 µL of 2 M H₂SO₄ per well was used to stop the chromogenic reaction and an ELISA reader was used to measure the absorbance values at 450 nm.