Pre-treatment of fish sera and antibody detection by ELISA were
carried out according to methods described elsewhere [25e28].
Two ELISA plates (Corning) were prepared with 50 mL of viral antigens
(VHSV or IHNV), which were diluted 1:10 with distilled
water and incubated at 37 C overnight. After 3 washes with
phosphate-buffered saline (PBS, pH 7.5) containing 0.05% Tween 20
(T-PBS), the plates were blocked with 5% skim milk in PBS at 25 C
for 1 h and washed again 3 times with T-PBS. Fish sera were diluted
40-fold with PBS containing 5% skim milk and incubated with 5%
skim milk at 25 C for 1 h. Another set of fish sera was diluted with
5% skim milk supplemented with 50% FBS.
P