O phenylenediamine dihydrochloride substrate reagent

The presence of TNP-specific IgM in serum was estimated by ELISA as previously described (27 (link)). For this, microtitre plates were coated with 5 μg/ml of TNP-BSA (Biosearch technologies) in a volume of 100 μl PBS overnight at 4°C. Thereafter, non-specific binding sites were blocked by incubation with 1% BSA in PBS with 0.05% Tween 20 (PBT) for 1 h at RT. Plates were then washed with PBT and a 1/500 dilution of each serum sample in PBS 1% BSA added to each well and incubated for 1 h at RT. Serum samples from all groups were analyzed in duplicate wells. After washing three times with PBT, each well was incubated with 1 μg/ml biotinyilated anti-trout IgM mAb (clone 4C10) diluted in PBS 1% BSA for 1 h at RT. The plates were washed again three times in PBT and 100 ng/ml of HRP-streptavidin (Thermo Fisher Scientific) added to each well in 100 μl PBS 1% BSA. After incubation at RT for 1 h, 100 μl of o-phenylenediamine dihydrochloride substrate reagent (Sigma-Aldrich) were added to each well. The reaction was stopped after 15 min by adding 50 μl of 2.5 M H₂SO₄. Absorbances were recorded at 490 nm using a FLUOstar Omega (BMG Labtech) plate reader. Internal positive and negative control samples were also included.

The presence of IPNV-specific trout IgM in serum of immunized fish was estimated by ELISA following a protocol previously described by Munang’andu et al. (29 (link)). For this, plates were coated with 100 μl of polyclonal anti-IPNV antibody diluted at 1:5000 in diluent buffer (0.05 M carbonate buffer pH 9.7) and incubated overnight at 4°C. Plates were washed 3 times in PBS-0.05% Tween-20 (PBT) and blocked with 5% fat free dry milk-PBS for 2 h at RT. Viral antigen of the T_T217A₂₂₁ IPNV strain was added to each well after incubation at RT for 2 h. Diluted sera in 1% dry milk 5% fat free dry milk-PBS were added to each well. Blank and positive controls were also included. After incubation at 4°C overnight, each well was incubated with 1 μg/ml biotinyilated anti-trout IgM mAb (clone 1.14) diluted in 5% fat free dry milk-PBS for 1 h at RT. The plates were washed again three times in PBT and 100 ng/ml of HRP-streptavidin (Thermo Fisher Scientific) added to each well in 100 μl 5% fat free dry milk-PBS. After incubation at RT for 1 h, 100 μl of o-phenylenediamine dihydrochloride substrate reagent (Sigma-Aldrich) were added to each well. The reaction was stopped after 15 min by adding 50 μl of 2.5 M H₂SO₄. Absorbances were recorded at 490 nm using a FLUOstar Omega (BMG Labtech) plate reader. Internal positive and negative control samples were included in all assays.