Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species

Escandon, Paulina; Heatley, J Jill; Berghman, Luc R; Tizard, Ian; Musser, Jeffrey MB

Western blot assays were performed according to Guo et al (2014) , 27 with the following modifications. Recombinant N-protein was separated using SDS-PAGE and the protein was electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated in 5% dried skim milk/0.05%Tween/0.02M PBS overnight at room temperature. The membrane was then incubated for 2 hrs with plasma that had been diluted 1:200 with 3 mL of 2% dried skim milk/0.02M PBS/ 0.05%Tween, and followed with three 15 min washes of PBS/0.05% Tween. The membrane was then incubated for 1 hr with one of the four conjugated secondary antibodies. The anti-macaw and anti-bird IgY secondary antibodies, which had initial concentrations of 1.0 mg/mL, were diluted 1:50,000 with 3 mL of 2% dried skim milk/PBS/ 0.05% Tween; the anti-chicken and anti-duck IgY secondary antibodies, which had initial concentrations of 0.1 mg/ mL, were diluted 1:5,000 with 3 mL of 2% dried skim milk/PBS/0.05% Tween. This was followed by three 15 min washes with PBS/0.05%Tween. The membrane was then incubated for approximately 5 mins, or until color change was observed, in a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) solution (SigmaFast BCIP/NBT substrate tablet, Sigma Aldrich, St. Louis, MO. USA) dissolved in 10 mL ultrapure water. Finally, the membrane was rinsed with ultrapure water.