The kinetics of chicken IgY anti-STAg production and avidity maturation were evaluated by an indirect ELISA. The optimal conditions for ELISA were obtained through block titration of the reagents. Briefly, high affinity microtiter plates (Costar Corning Incorporated, USA) were coated with STAg (10 mg/mL) in 0.06 M carbonate buffer (pH 9.6) and incubated overnight at 4uC. Plates were washed 3 times with PBS-Tween 0.05% (PBS-T) and blocked with PBS-T plus 1% bovine serum albumin (PBS-T-BSA) for 1 h at room temperature. P2 samples were adjusted to 2 mg/well in PBS-T-BSA, added to the wells in duplicate and incubated for 1 h at 37uC. After washing, plates were incubated with rabbit anti-IgY antibody labeled with peroxidase, diluted 1:30.000 in PBS-T, for 1 h at 37uC. The reaction was revealed by adding 0.01 M 2,29-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS, Sigma) and 0.03% H 2 O 2 , and optical density (OD) was determined at 405 nm. Two positive quality-controls and six negative controls (irrelevant IgY) were included in each plate in order to calculate the cut off, which was established as the mean OD values for irrelevant IgY plus three standard deviations. The same protocol was applied to mouse IgG anti-STAg obtained after immunization, using a goat IgG anti-mouse IgG conjugated to peroxidase (Sigma), at 1:2000 dilution. Results were expressed as ELISA index (EI) as previously proposed [47] as follows: EI = OD sample /OD cut off , where values of EI .1.2 were considered positive.