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)).
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)).
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