Total antibody titers against sH5 3 were determined by using a sH5 3 -specific ELISA. To this end, 96-well Nunc MaxiSorp plates coated with 0.5 mg sH5 3 per well were incubated with limiting dilutions of chicken or mouse sera. After extensive washing, the plates were incubated with goat-anti-chicken or goat-anti-mouse antibodies conjugated with HRP. Peroxidase activity was visualized using tetramethylbenzidine substrate (BioFX) and an ELISA reader (EL-808 [BioTEK]), reading the OD at 450 nm.