Trout IgM ELISA Protocol

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.