Immunoblotting of Trout Ig Isotypes

Buccal mucus and serum samples were resolved on 4–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing conditions as described previously (13 (link), 19 (link)). For western blot analysis, the gels were transferred onto PVDF membranes (Bio-Rad). Thereafter, the membranes were blocked with 8% skim milk and incubated with anti-trout IgT (rabbit polyclone antibody, pAb), anti-trout IgM (mouse monoclonal antibody, mAb), or biotinylated anti-trout IgD (mouse mAb) antibodies followed by incubating with peroxidase-conjugated anti-rabbit, anti-mouse IgG (Invitrogen) or streptavidin (Invitrogen). Immunoreactivity was detected with an enhanced chemiluminescent reagent (Advansta) and scanned by GE Amersham Imager 600 Imaging System (GE Healthcare). The captured gel images were analyzed by ImageQuant TL software (GE Healthcare). Thereafter, the concentration of IgT, IgM, and IgD were determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified trout IgT, IgM, or IgD.