P0159

Protein samples were resolved on a 12% SDS-PAGE gel and transferred to nitrocellulose membranes in a semi-dry electrophoretic transfer apparatus (BioRad, Temse, Belgium) using transfer buffer (3.03 g Tris-base, 14.41 g Glycine and 200 mL Methanol in final volume of 1 L) at 100 V for 1 h. Following the protein transfer onto membranes, the blocking was performed overnight at 4 °C in buffer containing 5 mM Tris HCL pH 8, 15 mM NaCl, 0.05% Tween-20 (TBS-T) and 5% bovine serum albumin (BSA). After blocking, washing was performed with TBS-T. Next, the membranes were incubated with cattle serum 1:200 as primary antibody for 1 h in 5% BSA in TBS-T. After three wash cycles with TBS-T, membranes were incubated with 1:6000 HRP conjugated rabbit anti cow antibody (P0159, DAKO, Heverlee, Belgium) for 1 h with 0.1% BSA in TBS-T. Following incubation, membranes were washed three times with TBS-T. The proteins were visualized using an ECL chemiluminescent substrate reagent kit (Thermo Fischer Scientific, Brussels, Belgium) according to manufacturer’s instructions.

To check the efficacy of bovine sera to recognize the NS4B protein, an indirect ELISA was performed as described elsewhere [19 (link)]. In short, each well of microtiter plate was coated overnight at 4 °C with 300 ng/well with the membrane protein fractions containing NS4B in 0.1 M carbonate buffer pH 9.6. Lysate of MDBK-BVDV-NADL was used as positive control. After each step, washing was performed five times with washing buffer (PBS, 0.05% Tween-20). Blocking was performed at 37 °C for 1 h with PBS, 0.05% Tween-20, 5% horse serum (PBS-T-HS), 5% skimmed milk powder. Following the addition of 100 μL of diluted bovine serum at 1:200 in PBS-T-HS in each well, incubation was performed at 37 °C for 1 h. Next, 100 μL of 1:5000 HRP conjugated rabbit anti cow antibody (P0159, DAKO, Heverlee, Belgium) in PBS-T-HS was added was applied for 1 h at 37 °C. Finally, ABTS substrate (Thermos Fischer Scientific, Brussels, Belgium) solution was added and absorbance was measured at 405 nm after incubation for 20 min at room temperature.