3 n h2so4

MaxiSorp plates (Nunc, Rochester, NY, USA) were coated with NS1 purified baculovirus expressed proteins (164 ng per well) and incubated overnight at 4 °C. Plates were saturated with blocking buffer (PBS 0.05% Tween 20 and 5% skim milk). The animal sera diluted in blocking buffer were added and incubated for 1 h at 37 °C. After three washes in PBS—0.05% Tween 20, plates were incubated for 1 h at 37 °C with an anti-mouse-HRP secondary antibody (BioRad, Portland, ME, USA) at a 1/2000 dilution in blocking buffer. Finally, after three washes in PBS—0.05% Tween 20, the reaction was developed with 50 µL of substrate solution 3,3′, 5,5′–tetramethylbencidine liquidsupersensitive (TMB) (Sigma Aldrich, St. Louis, MO, USA) and stopped by adding 50 µL of 3 N H₂SO₄ (Merck KGaA, Darmstadt, Germany). Results were expressed as optical densities (ODs) measured at 450 nm.

MaxiSorp plates (Nunc) (Thermo Fisher Scientific, NY, USA) were coated with NS1 (250 ng per well) or NS2-Nt (200 ng per well) purified baculovirus expressed proteins in PBS and incubated overnight at 4°C. Plates were saturated with blocking buffer (PBS-0.05%-Tween 20-5% skim milk). Individual mice or sheep sera diluted in blocking buffer were added and incubated for 2 h at 37°C. After three washes in PBS-0.05% Tween 20, plates were incubated for 1 h at 37°C with an anti-mouse-HRP secondary antibody (Bio-Rad, Hercules, CA, USA) (1:2,000) or with an anti-sheep-HRP secondary antibody (Sigma-Aldrich, San Louis, MO, USA) (1:7,500) in blocking buffer. Finally, after three washes in PBS-0.05% Tween 20, the reaction was developed with 50 μL of TMB (Thermo Fisher Scientific, MD, USA) and stopped by adding 50 μL of 3 N H₂SO₄ (Merck, Darmstadt, Germany). Results were expressed as optical densities (ODs) measured at 450 nm.