The level of anti-betanodavirus antibodies in sera from immunized sole have been evaluated following the indirect ELISA procedure previously reported (65 (link)). Briefly, sera from sole immunized with the different recombinant viruses (20 µg of total proteins) were diluted in coating buffer [100 mM Bicarbonate/Carbonate, pH 9.6] and immobilized in 96 High Binding flat-bottomed plates (Sarsted, Newton, NC, USA) overnight at 4°C. The samples were blocked with 5% skimmed milk in PBST for 1 h. Afterwards, incubation with a rabbit anti-NNV (484.2.2009, 1:10,000) was performed for 1 h at room temperature. Following washing steps, the samples were incubated with the anti-rabbit IgG-HRP (Sigma Aldrich; 1:25,000) for 1 h at RT. The reaction was revealed with 100 µL per well of 3,3’,5,5’-tetramethylbenzidine single solution (ThermoFisher, Waltham, MA, USA) for 20 min and stopped by adding 50 µL of 2 M sulfuric acid. Optical density (OD) was measured at 450 nm. Resulting OD values were normalized by subtracting the OD values of the negative control (omitting fish sera) wells. All assays were performed in duplicate and previously assayed positive serum was used as a positive control.
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