El311sx

Sucrose gradient-purified viruses were diluted in PBS and added to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 hemagglutinating units (HAU) per well. After incubation overnight at 4 °C, samples in wells were blocked with PBS-nonfat dry milk. Antisera against the F/98 virus in chicken were then added in serial twofold dilutions with PBS containing 0.05% Tween-20, and incubated for 3 h at 37 °C. After washing, goat anti-chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and allowed to incubate for 1.5 h at 37 °C. After washing, TMB (3,3′,5,5′-Tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H₂SO₄. Absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT) [22 (link)].

To assay antibodies in catfish serum and skin mucus, ELISA was performed using E. piscicida LPS following the protocol previously described [29 (link)]. In brief, E. piscicida LPS (100 ng/well) in coating buffer was used to coat the polystyrene 96-well flat-bottom microtiter plates (Dynatech Laboratories Inc., Chantilly, VA, USA). Wells were blocked by adding 300 μL of 5% bovine serum albumin (BSA). Diluted samples of catfish serum or 100 μL of mucus was added to wells in duplicate, while in blank control wells, 100 µL of sterile PBS was added. Moreover, 100 μL of diluted mouse anti-catfish IgM monoclonal antibody (Aquatic Diagnostics Ltd., Scotland, UK) was added to each well. Biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL, USA) was diluted, and 100 μL of it was added to the wells. For the color development, p-nitrophenyl phosphate (PNPP, Thermo Fisher Scientific, Rockford, IL, USA) was added, and the OD was noted at 405 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT, USA).

ELISA was used to assay antibodies in gill, skin and serum to E. piscicida LPS and Ich membrane protein. Samples were prepared as described previously (5 (link)). Polystyrene 96-well flat-bottom microtiter plates (Dynatech Laboratories Inc., Chantilly, Va.) were coated with E. piscicida LPS or Ich membrane protein (100 ng/well), in sodium carbonate-bicarbonate coating buffer (pH 9.6) 100 μl volumes in each well. The coated plates were incubated for an overnight at 4°C. Free binding sites were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. After washing, 100-μl volume of diluted zebrafish anti-serum/mucus sample was added to individual wells in duplicate and incubated for 2 h at 37°C. The plates were treated with mouse anti-zebrafish IgM monoclonal antibody (Aquatic Diagnostics Ltd) for 1 h at room temperature. Plates were then incubated with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) for 1 h at room temperature. After incubation of wells with a streptavidin-alkaline phosphatase conjugate (Southern Biotechnology) for 1 h at 37°C, p-nitrophenyl phosphate (PNPP, Thermo Fisher Scientific) was added for color development. The optical density (OD) units were read at 405 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT).