*2.6. ELISA Procedure*

The operation procedure of indirect ELISA (i-ELISA) described in Peng et al. was used with some modifications [26]. Microculture plates were added PBA-OVA coating antigen (100 μL/well), incubated at 4 ◦C overnight. After being three times washed by PBST (0.05% Tween 20 in PBS, pH 7.4), 200μL of OVA-PBS (1% OVA in PBS) was added into every well and blocked for 1 h at 37 ◦C. Washed again, 100 μL antibody was added into every well, and the plates kept for 40 min at 37 ◦C. Washed again, HRP-IgG (1:6000 dilution in 0.1M PBS, 100 μL/well) was added to all wells and the plates was kept at 37 ◦C for 40min. After being washed four times, TMB substrate solution (100 μL/well) was added. After incubation at 37 ◦C in the dark for 15 min, 50 μL of 1 M H2SO4 was added to finish the reaction. The absorbance at 450 nm was determined by an automatic ELISA reader.

The operation procedure of indirect competitive ELISA (ic-ELISA) was in common with i-ELISA. The only difference was that the mAb and drug standard or sample (50 μL/well) were added to each well. The standard curve was established with logarithm of analyte concentration as the abscissa and the inhibition values (B/B0) corresponding to each concentration as the ordinate.
