A 2D-immunoblot assay was also carried out to evaluate the antigen recognition profile of immunized hens to STAg. Briefly, 60 mg of STAg dialysed in ultrapure water, was separated by isoelectric focusing (IEF) on 7-cm immobilized pH gradient strips (ReadyStrip TM IPG Strip pH 3-10) overnight at room temperature, following the manufacturer instructions for equipment and reagents (GE, Healthcare, Uppsala, Sweden). After IEF, strips were equilibrated and loaded onto precast 12% polyacrylamide gels. Electrophoresis was performed and 2D-gels were stained with Coomassie brilliant blue G-250H (Sigma) or electrotransferred to nitrocellulose membranes. 2D-immunoblot was performed as described above for 1D-immunoblot.