Hrp goat anti mouse igm

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.

For the detection of α-Gal in the glycoproteins of T. canis (Tc-ExAD), A. suum (As-ExAD) and the larval ES products of T. canis (Tc-ES), the parasites’ soluble antigens (1 μg/mL or 100 ng/well) were coated overnight at 4 °C on a microplate (Nunc-Immuno^TM Plate, Roskilde, Denmark). Galα1-3Gal-HSA antigen (Dextra Laboratories, Reading, UK) served as a positive control (0.5 μg/mL or 50 ng/well). After blocking with 1% HSA (1 h/37 °C) and washing with PBS-T, the presence of α-Gal epitopes in the protein extracts was evaluated by the M86 mAb (Enzo LifeScience Inc., Farmingdale, NY, USA) diluted at 1:400. HRP-goat anti-mouse IgM (Bio-Rad Laboratories Inc., Feldkirchen, Germany) was used at a 1:4000 dilution as a secondary Ab, and the following ELISA steps were performed as described above.