Wells were coated with 100 μl of antigen (10 mg of OVA/ml of phosphate-buffered saline-PBS) at 4 °C for 12 h, washed and incubated with 1% skimmed milk (200 μl) at 37 °C for 1 h to reduce non-specific binding. After washing, the plasma (1:5000 dilution in PBS; 100 μl per well) was added and incubated at 37 °C for 1 h. The extent of antibody binding was detected using a horseradish
peroxidase-conjugated donkey anti-bovine IgG (Sigma Chemical Co., Italia) (1:20,000 dilution in PBS; 100 μl per well).