Enhanced chemiluminescent kit

Soluble crude proteins from diverse T. spiralis phase worms, 6-h IIL ES proteins, and rTsGS were separated by 12% SDS-PAGE (Wang et al., 2013 (link); Ren et al., 2018 (link)). The proteins were transferred onto a nitrocellulose membrane (Millipore, United States). The membrane was blocked with 5% skimmed milk diluted in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20 (TBST) at 37°C for 2 h and cut into strips. The strips were probed with 1:100 dilutions of various sera (anti-rTsGS serum, infection serum, and normal serum) at 37°C for 2 h. After washing with TBST, the strips were incubated with HRP-conjugated anti-mouse IgG (1:10,000; Southern Biotech, United States) at 37°C for 1 h. After washing again, the strips were developed with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) or using an enhanced chemiluminescent kit (CWBIO, Beijing, China) and terminated by washing the membrane with deionized water (Long et al., 2014 (link); Liu et al., 2018 (link)).

Soluble somatic proteins of various T. spiralis stages (ML, IIL, 3 dpi AW and NBL) and rTsAP were identified by western blotting with anti-rTsAP serum. Worm proteins and rTsAP were separated by SDS-PAGE, then transferred onto nitrocellulose membrane (Merck Millipore, MA, USA) at 18 V for 35 min [34 (link)]. The membrane was cut into strips that were blocked using 5% skimmed milk in TBST at 37 °C for 2 h. Following washing with TBST, the strips were reacted with anti-rTsAP serum (1:100) for 1 h at 37 °C, and followed by incubation of HRP-conjugated anti-mouse IgG (1:10,000; Sigma-Aldrich, USA) at 37 °C for 1 h. After washing again, the strips were developed with 3, 3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich), and finished by washing the membrane with deionized water [21 (link), 35 (link)].
To ascertain the relative TsAP protein expression level at various T. spiralis stages, 15 μg/lane of soluble proteins of ML, IIL, 3 dpi AW and NBL was analyzed using SDS-PAGE and western blot with 1:100 dilutions of anti-rTsAP serum [36 ]. An antibody against GAPDH (1:1000) was used to assess GAPDH expression as an internal quantitative control [37 (link)]. After washing, the strip was colored with an enhanced chemiluminescent kit (CWBIO, Beijing, China) [29 (link)]. The relative TsAP protein expression levels in T. spiralis different stages were analyzed by Image J software.

Soluble crude antigens of T. spiralis obtained at various stages, IIL ES antigens and purified rTsGSCP were separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose (NC) membranes (Millipore, USA) in semidry transfer cells (Bio-Rad, USA) [37 (link), 48 (link)]. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween (TBST) at 37 °C for 2 h and cut into strips. The strips were probed by various sera (1:200; anti-rTsGSCP serum, infection serum and preimmune serum) at 37 °C for 2 h. Following washes with TBST, the strips were incubated at 37 °C for 1 h with HRP-conjugated anti-mouse IgG (1:10 000; Southern Biotech, USA). After being rewashed, the strips were coloured using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, USA) or reagent from an enhanced chemiluminescent kit (CWBIO, Beijing, China) [49 (link)].