3 3 5 5 tetramethylbenzidine single solution

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.

Serum anti-dsDNA antibody levels were measured by ELISA. The optical density at 450 nm was read using a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Waltham, MA, USA). Serum was diluted 1:100 in phosphate-buffered saline (PBS), and then diluted two-fold seven times. ELISA plates were pre-treated with polylysine (2 μg/mL) at 37 °C for 2 h. The plate was then washed with PBS containing 0.1% Tween 20 three times (PBST). After washing, ELISA plates were coated with mouse dsDNA solution (5 μg/mL) at 4 °C overnight. Afterward, the plates were washed and blocked with 5% fetal bovine serum at 37 °C for 2 h. After washing, serial dilutions of test sera were added to each well and incubated at 37 °C for 1.5 h. The plates were washed three times with PBST and incubated with horseradish peroxidase-conjugated goat-anti-mouse IgG (Sigma–Aldrich, MO, USA) at 37 °C for 45 min. After washing the plates three times, 3,3′,5,5′-tetramethylbenzidine single solution (Thermo Fisher Scientific) was added to each well and the colorimetric reaction was allowed to develop at room temperature for 3 min. The reaction was stopped with 2 M H₂SO₄ and then the optical density was determined.