To investigate the antigen recognition repertoire of IgY anti-STAg antibodies, immunoblot assays were carried out along with avidity maturation analysis against STAg. In 1D-immunoblot, STAg was separated on 12% SDS-PAGE under non-reducing conditions, and electrotransferred to nitrocellulose membranes. Non-specific interactions were blocked by 5% skim milk-PBS-T incubation, for 2 h at room temperature. Nitrocellulose strips were then incubated with P2 samples from weekly egg yolk pools adjusted to 2 mg of IgY. In parallel, avidity maturation was measured by strip treatment with 6 M urea for 10 min at room temperature. IgY was detected by incubating the secondary antibody, rabbit anti-IgY labelled with peroxidase, diluted at 1:20.000, for 2 h at room temperature. Reaction was revealed by adding 10 mg of 3,39-diaminobenzidine tetrahydrochloride (DAB, Sigma) in 15 mL of Tris buffered saline (TBS) and 12 mL of 30% hydrogen peroxide (Sigma). Reaction was stopped with distilled water. Experiments with similar conditions were performed for IgY anti-N. caninum and anti-Eimeria spp., as well as for the establishment of recognition kinetics of mouse IgG obtained from mice immunized by i.m. and s.c. routes (1:100 sera dilution; 1:2000 anti-mouse IgG conjugated to peroxidase -Sigma).