ELISA for Detecting Antibodies Against nvIBDV

The 96-well plates (One Riverfront Plaza Corning, Corning, NY, USA) were coated with 100 ng per well of purified nvIBDV VP2 protein expressed by Pichia pastoris (P. pastoris) (Ruipu, Tianjin, China) in the coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) at 4 °C overnight and then blocked by 5% non-fat milk at room temperature for 1 h [25 (link)]. After that, 100 μL of 1:500 diluted chicken sera was added to each well of the coated plates. The plates were incubated at 37 °C for 30 min, followed by washing with PBST three times. The goat anti-chicken IgY secondary antibody HRP (Invitrogen, Corning, NY, USA) diluted with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05% Tween-20, pH 7.4) was added, and the plates were incubated at 37 °C for another 30 min. After washing three times with PBST, the plates were visualized by adding 100 μL 3,3′,5,5′-tetramethylbenzidine solution (Tiangen, Beijing) and incubated for 10 min at room temperature. Reactions were terminated by adding 50 μL of 2 M sulfuric acid. Absorbance at 450 nm was recorded using an Eon™ High Performance Microplate Spectrophotometer (BioTek, Shoreline, WA, USA). The S/P value was calculated by the following formula: (S/P value = (sample value − mean value of NC)/(mean value of PC − mean value of NC)). A sera sample with an S/P value > 0.2 was considered positive.