Evaluation of reactivity of bovine mAbs with the 146S and 12S particles of A/AF72 strain (A) and A/GDMM/2013 strain (B) using indirect ELISA.

12S particles were prepared from naïve 146S particles by acidification (incubation with NaH2PO4 (pH = 5.5) for 10 mins) or heat treatment for 1 h at 56°C. In indirect ELISA experiments, 100 ng/well of naïve 146S / acid treated 146S / heat treated 146S was respectively coated in 96-well plates overnight at room temperature. The plates were then washed three times with PBST (PBS buffer plus 0.05% Tween) and blocked with 1% gelatin in PBS at 37°C for 2 h. After three washes, the bovine mAbs at different concentrations were added and incubated at 37°C for 1 h. The plates were washed three times with PBST, and then the HRP-conjugated anti-His tag antibody (Genscript, China) at a dilution of 1:5,000 was added to the wells. The plates were then incubated at 37°C for 30 min and washed three times with PBST. Color was developed by adding 50 μl of TMB substrate (Pierce, Life Technology) for 10 min at room temperature. The process was stopped by adding equal volumes of 1 M H2SO4. Optical density at 450 nm (OD450) was measured on a microplate reader (BioRad). The results represent one of three independent assays with duplication. (TIF)