Anti c1q mab

To further demonstrate the interaction between rTs-Pmy and complement C1q, purified human C1q (5 μg; Merck, Germany) under reducing condition were subjected to SDS-PAGE using 12% polyacrylamide gel followed by either Coomassie bule staining or Far Western blotting. After blocking with 5% dry milk in 1 × PBS, the membrane was incubated with rTs-Pmy (5 μg/ml in PBST and 1% dry milk) at 37°C for 2 h and then washed with PBST. The membrane was probed with an anti-His mAb (rTs-Pmy contains His-tag) (Tiangen, China; 1:5,000 in PBST containing 1% dry milk) for 1 h at room temperature. IRDye 800 CW-labeled goat anti-mouse IgG (LI-COR, Germany; 1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Vice versa, rTs-Pmy, non-relevant control BSA and rTs87 (5 μg each) were subjected to 12% SDS-PAGE and then transferred to a NC membrane. After blocking, the membrane was probed with human complement C1q (5 μg/ml in PBST containing 1% dry milk) at 37°C for 2 h. The anti-C1q mAb (Abcam, USA; 1:1,000 in PBST containing 1% dry milk) was used as the primary antibody and IRDye 800 CW-labeled goat anti-mouse IgG (1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Membranes were visualized and imaged using an Odyssey infrared imaging system (LI-COR, Germany).

To verify whether native Ts-Pmy from T. spiralis was able to bind to C1q, Protein G Micro Beads (Miltenyi Biotec, Germany) were pre-incubated with the anti-Ts-Pmy mAb 9G3 (2 μg) and T. spiralis adult worm crude extracts (40 μg) for 30 min on ice. Then, human complement C1q (3 μg) was added and the incubation was continued for 2 h at 4°C. The beads were washed four times with washing buffer (1% NP40 substitute, 50 mM Tris buffer, pH 8.0), and the bound proteins were eluted in 1× SDS gel loading buffer by boiling for 5 min. The eluted proteins were subjected to 12% SDS-PAGE and transferred to a NC membrane. The membrane was probed with an anti-C1q mAb (Abcam, USA) at a 1:1,000 dilution in PBST containing 1% dry milk; IRDye 800CW-labeled goat anti-mouse IgG (1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody.