Hrp conjugated goat anti mouse igg h l antibody

Antibody levels of PmCQ2Δ4555–4580 were determined as the method described previously [32 (link)]. Briefly, total bacterial cell proteins of PmCQ2Δ4555–4580 were prepared by using ultrasound pyrolysis, and the proteins concentration was measured by Bradford method. Each well of 96-well ELISA plates was coated with 1 µg protein in 100 µL carbonate buffer (0.05 M, pH9.0) at 4 °C overnight. The next day, the plates were washed 5 times with PBST (PBS containing 0.05% Tween-20), and then treated with blocking buffer (5% skim milk in PBST) at 37 °C for 1 h. Then, the sera were serially diluted in twofold increments in 96-well plates and incubated at 37 °C for 1 h. Next, 100 µL of HRP-conjugated goat anti-mouse IgG (H + L) antibody (Sigma; diluted at 1: 10 000) was added and incubated for 1 h at 37 °C. Then, 100 µL of TMB were added for 10 min (Beyotime biotechnology, China) and stopped by the addition of 2 M H2SO4, before the absorbance quantification at OD₄₅₀ was done. When the ratio of the positive value (P) of the maximum dilution multiple sera of immunized mice to the negative value (N) of sera of non-immunized mice is greater than 2.1 (P/N > 2.1), the maximum dilution ratio is the serum antibody titer.

Total bacterial cell proteins of each strain were prepared by using ultrasound pyrolysis and the proteins concentration was measured by using the Bradford method. Each well of 96-well ELISA plates was coated with 1 μg protein in 100 μL carbonate buffer (0.05 M, pH9.0) at 4 °C overnight. The next day, the plates were washed 5 times with PBST (PBS containing 0.05% Tween-20), and then treated with blocking buffer (5% skim milk in PBST) at 37 °C for 1 h. Next, the plates were washed 5 times with PBST. The sera were serially diluted in twofold increments in 96-well plates and incubated at 37 °C for 1 h. The sera from non-immunized mice served as negative controls. After washing, 100 μL of HRP-conjugated goat anti-mouse IgG (H + L) antibody (Sigma; diluted at 1: 10 000) was added and incubated for 1 h at 37 °C, followed by washing. Then, 100 μL of TMB were added for 10 min (Beyotime biotechnology, China) and stopped by the addition of 2 M H₂SO₄, before the absorbance quantification at OD₄₅₀ was done [25 (link)]. When the ratio of the positive value (P) of the maximum dilution multiple sera of immunized mice to the negative value (N) of sera of non-immunized mice is greater than 2.1 (P/N > 2.1), the maximum dilution ratio is the serum antibody titer.