Galα1 3gal hsa antigen

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.

To assess the specificity of anti-α-Gal Abs in turkeys, the Galα1-3Gal-HSA antigen (Dextra Laboratories, Reading, UK) was immobilized on an ELISA plate (50 ng/well), and treated or not with α-galactosidase from green coffee beans (Sigma-Aldrich, St. Louis, MO, USA), following the procedure described elsewhere [36 (link)]. Before the treatment, the enzyme was centrifuged at 10,000× g for 10 min at 4 °C, to remove the ammonium sulfate. The supernatant was discarded and 100 mM potassium phosphate buffer (pH 6.5) was added to the pellet, so the final concentration of the enzyme solution was 50 mU/100 µL. The plate was then incubated at 37 °C for 24 h in a humidified plastic chamber to avoid evaporation. After the incubation, wells were washed five times with 150 µL of PBST and the indirect ELISA was performed as described above. Sera samples from the turkeys treated with E. coli O86:B7 (n = 5), E. coli BL21 (n = 5) and PBS (n = 5) were randomly selected and used in the specificity assay. The statistical differences of sera reactivity against treated and non-treated antigen were evaluated using the Wilcoxon signed rank test in the GraphPad 5 Prism program (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant when p < 0.05.

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.