Antibody Titer Determination of PmCQ2Δ4555–4580

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.