Biotinylated goat anti mouse igg

Ninety-six-well flat plates were coated with either salmon sperm DNA (Sigma) or with goat anti-mouse IgM, IgA, IgE, IgG1, IgG2b (Southern Biotech). After washes, they were saturated with BSA and first incubated with mice sera, then with biotinylated goat anti-mouse IgG (Southern Biotech) or goat anti-mouse IgM, IgA, IgE, IgG1, IgG2b (Southern Biotech). A streptavidin-horseradish conjugate (Sigma) was added followed by the addition of TMB (eBioscience). The reaction was stopped with HCl (1N) and revealed with an ELISA plate reader DTX880 Multimode Detector (Beckman Coulter).

To determine antibody titers in murine serum, HiBond ELISA plates were coated with whole GAS-M1 bacteria, or recombinant M1 protein or isolated M1 HVR [generated as described in (38 (link))] and incubated with serially diluted serum. Captured antibodies were detected using biotinylated goat anti-mouse IgG (Southern Biotech, CAT 1036-08), IgG1 (BioLegend, RMG1-1), IgG2b (BioLegend, RMG2b-1), and IgG2c (Southern Biotech, CAT 1079-08) and streptavidin-HPR (BioLegend, CAT 405210). BSA-coated wells served as negative controls and presented values represent the average of duplicate experimental values with deducted negative control values. All samples were run in duplicate.

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).

Luminex assays were performed as reported^{39 (link)}. In brief, 5 × 10⁶ microspheres (Luminex Corp.) were covalently linked to 25 μg of recombinant protein and incubated with serially diluted mAb (mouse IgG1). Bound mAbs were detected with 4 μg/ml biotinylated goat anti–mouse IgG (SouthernBiotech) (SouthernBiotech), followed by incubation with 5 μg/ml streptavidin-PE (BD). Fluorescence was measured on a Bio-Plex instrument (Bio-Rad).