Briefly, 15 µg of the adult worm crude extracts (Tc-ExAD, As-ExAD) and Tc-ES antigens were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following the protocol [35 (link)]. After electrophoresis, the resolved proteins were transferred to nitrocellulose membranes (Trans-Blot-Turbo-Transfer-System, Bio-Rad Laboratories Inc., Feldkirchen, Germany). The membranes were blocked with 1× casein solution (Vector Laboratories Inc., Burlingame, CA, USA) and incubated with a monoclonal mouse anti-α-Gal antibody (mAb) M86 (Enzo LifeScience Inc., Farmingdale, NY, USA) diluted 1:5 in Tris buffered saline buffer (TTBS; 100 mM Tris, 0.9% NaCl, 0.1% Tween 20). After rinsing in TTBS buffer for 15 min (3 × 5 min), blots were incubated with HRP-goat anti-mouse IgM (Bio-Rad Laboratories Inc., Feldkirchen, Germany), diluted 1:300 in TTBS. All the incubation steps were carried out at room temperature. Immunoreactive proteins were detected by using the DAB-peroxidase substrate kit (Vector Laboratories Inc., Burlingame, CA, USA) and following the manufacturer’s instructions. Galα1-3Gal-HSA antigen (5 µg; Dextra Laboratories, Reading, UK) was used as a positive control.
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