Hrp conjugated anti mouse igg

The purified rTsCTL were separated on 10% SDS-PAGE [35 (link)]. The proteins were transferred onto nitrocellulose (NC) membrane (Millipore, USA) in the wet transfer cell (Bio-Rad, USA) [36 (link)]. The membrane was blocked using 5% skimmed milk in Tris-buffered saline containing 0.05% Tween (TBST) at 37 °C for 2 h, and cut into strips. The strips were incubated with diverse serum (1:100; anti-rTsCTL serum, infection serum, anti-GST serum and normal serum) at 37 °C for 2 h. After being washed using TBST, the strips were incubated at 37 °C for 1 h with HRP conjugated-anti-mouse IgG (1:10 000; Southern Biotech, USA). After washes, the color was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) [37 (link), 38 (link)].

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)).

Sera and BAL from different time-points were screened for humoral response against SARS-CoV-2 spike. To measure IgG and IgA antibody levels in the plasma and BAL of mice, respectively, a SARS-CoV-2-specific enzyme-linked immunosorbent assay (ELISA) was developed. Briefly, Nunc ELISA plates were coated with SARS-CoV-2 spike protein (BEI resources-NR-52396, 100 ng total/well) diluted in carbonate/bicarbonate buffer, pH 9.6, and incubated overnight at 4 °C followed by blocking with 5% skim milk to reduce background. A total of 100 μL of diluted serum (1/25) or BAL (undiluted) harvested at different time-points from immunized mice was added to the wells and incubated at 37 °C for 1 h. Post washing (PBS-TritonX 100, 0.1%), either HRP-conjugated anti-mouse IgG (1036-05, Southern Biotech, Birmingham, AL, USA) or anti-mouse IgA (1040-05, Southern Biotech) at dilutions of 1/1000 was added to the wells and incubated at 37 °C for 1 h. Post washing, 100 μL of TMB substrate solution was added and incubated for 20 min or until color developed. The addition of 1 M sulfuric acid stopped the reaction, and plates are read at 450 nm. Binding antibody end point titers (EPTs) were calculated as described previously [17 (link)].

Twenty mice were immunized subcutaneously with 20 μg rTsGS mixed with complete Freund’s adjuvant. Boost immunization was performed three times with 20 μg rTsGS mixed with incomplete Freund’s adjuvant at a 2-week interval (Zhang et al., 2020 (link)). At 2 weeks after the last immunization, the tail blood of immunized mice was collected to isolate anti-rTsGS immune sera (Cui et al., 2013b (link)).
The anti-rTsGS IgG level of all immunized mice was assessed by ELISA (Cui et al., 2015b (link)). Briefly, micro-plates were coated with 2 μg/ml rTsGS at 37°C for 2 h. After washing with phosphate-buffered saline (PBS; pH 7.4) containing 0.05% Tween-20, the plate was blocked with 5% skimmed milk at 37°C for 2 h. Following a wash, the diluted anti-rTsGS sera were added and incubated at 37°C for 2 h and then by incubation of HRP-conjugated anti-mouse IgG (1:10,000; Southern Biotech, United States) at 37°C for 1 h. Coloration was performed using OPD (Sigma-Aldrich) plus H₂O₂; the reaction was finished by the addition of 2 M H₂SO₄. The optical density values at 492 nm were measured by a microplate reader (Tecan Schweiz AG, Switzerland) (Liu L. N. et al., 2015 (link)).

96 well polyvinyl round bottom plates were coated with 20 μg/ml of affinity purified rabbit anti-rWb-Bhp-1 polyclonal antibody in 0.1M sodium carbonate buffer pH 8, incubated overnight at 37°C, then blocked with ELISA diluent at 37°C for 1 hour. Plates were coated with sera that had been boiled 5 min with an equal volume 0.1M EDTA pH 7.5 or with 500 μg/ml purified rWb-Bhp-1 boiled in 0.1M EDTA pH 7.5, an antigen that we have shown is heat stable. Plates were washed 5 times with PBST, incubated at 37°C for 1 hour with 1:1000 dilution of mouse anti-rWb-Bhp-1. Plates were washed 5 times with PBST, before HRP conjugated anti-mouse IgG (Southern Biotech) diluted at 1:4000 in ELISA diluent was added, and plates were incubated at 37°C for 1 hr. Plates were washed in PBST 5 times before adding the substrate o-phenylenediamine dihydrochloride. The enzymatic reaction was stopped with 4M H₂SO₄, and plates were read as described above.