The IgY concentrations in WSF were analyzed
using an in-house competitive ELISA. Microtiter
plates (Corning Inc., Lowell, MA) were coated with 10
ng of quail IgY/well (Alpha Diagnostic International,
San Antonio, TX) in 100 µL of coating buffer (0.05 M
carbonate buffer, pH 9.6). After incubation overnight
at 4°C in a moist chamber, the plates were blocked
with 2.5% casein (pH 7.3; 300 µL/well) for 2 h at room
temperature. The wells were washed 5 times with 300
µL of PBS and 0.05% Tween 20 (AppliChem GmbH,
Darmstadt, Germany) and were then stored at −20°C
until use.