Each well of the microtiter plates was first coated with 100 μL of the coating antigen in coating buffer and then incubated at 4°C overnight. Blocking buffer was then added to the plates (150 μL/well) and incubated for 2 h at 37°C. After washing, 50 μL of the analyte with serial dilutions was added to the wells as well as 50 μL of optimal diluted mAb. After incubation for 30 min at 37°C, the plates were washed two times and patted dry. Then, 100 μL of HRP labeled goat anti-mouse IgG (1:5000) was added, followed by incubation for 2 h at 37°C. After further washing, 100 μL of TMB substrate was added to each well, which was incubated for 15 min at 37°C. Finally, the chromogenic reaction was inhibited with 2 M H2SO4 (50 μL/well), and the OD values at 450 nm were measured.