Parasite products (ES products or somatic homogenate MvH) were prepared as described by Vendelova et al. [4 (link), 9 (link)], and the protein content was assessed using Bradford protein assay reagent (BioRad, Hercules, CA, USA) and using BSA (Sigma-Aldrich, St. Louis, USA) as the standard.
To determine the specific IgG/IgM, flat-bottom 96-well plates (Nunc Maxisorp) were coated with worm antigens (2.5 μg/ml ES or MvH) in carbonate-bicarbonate coating buffer (pH 9.6) per night at 4 °C. The microplates were blocked for 1 h at room temperature with 10% bovine fetal serum-PBS solution. Sera/peritoneal exudates diluted 1:100 were incubated for 1 h at 37 °C. The microplates were then washed, and goat anti-mouse IgG/IgM peroxidase conjugates diluted 1:5000/1:2000 (Sigma-Aldrich, St. Louis, USA) were added for 1 h at 37 °C. After further washes, O-phenylendiamine (Sigma-Aldrich, St. Louis, MO, USA) was added as a substrate, and absorbance values were recorded at 492 nm. Results were expressed as the mean optical density (OD) ± SD. The cut-off levels were determined as the mean + 3.8 × SD of the antibody activity in the exudates of healthy mice after Lardeux et al. [10 (link)].
To determine the specific IgG/IgM, flat-bottom 96-well plates (Nunc Maxisorp) were coated with worm antigens (2.5 μg/ml ES or MvH) in carbonate-bicarbonate coating buffer (pH 9.6) per night at 4 °C. The microplates were blocked for 1 h at room temperature with 10% bovine fetal serum-PBS solution. Sera/peritoneal exudates diluted 1:100 were incubated for 1 h at 37 °C. The microplates were then washed, and goat anti-mouse IgG/IgM peroxidase conjugates diluted 1:5000/1:2000 (Sigma-Aldrich, St. Louis, USA) were added for 1 h at 37 °C. After further washes, O-phenylendiamine (Sigma-Aldrich, St. Louis, MO, USA) was added as a substrate, and absorbance values were recorded at 492 nm. Results were expressed as the mean optical density (OD) ± SD. The cut-off levels were determined as the mean + 3.8 × SD of the antibody activity in the exudates of healthy mice after Lardeux et al. [10 (link)].
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