2.7. Serology

ELISA was performed to detect bovine immunoglobulin (IgG) according to the protocol described elsewhere [36,37]. Briefly, 10 µg/mL of native antigens were coated in a 96 well ELISA plate (BIOFIL^®, Guangzhou, China) in 50 mM bicarbonate buffer, incubated at 4 °C overnight. The ELISA plates were washed three times with washing buffer (0.05% Tween 20, 0.01 M PBS, PH 7.2). Saturation of the microtiter plate was performed with 4% BSA in PBS followed by incubation at 37 °C for 2 h. Both negative and positive sera were diluted in PBS to achieve two-fold serial dilutions. Diluted sera were poured into each well. The plate was incubated again at 37 °C for 1 h. The second washing was performed with a washing buffer as described above. Bound antibodies were detected by incubating at 37 °C for 2 h with goat anti-bovine IgG-alkaline phosphatase conjugate (1:10,000). After washing thrice, phosphatase activity was measured with P-nitrophenyl phosphate (pNPP, Cat. 41480004-1, Bioworld^®, Dublin, OH, USA) at 1 mg/mL in 1 M diethanolamine (Cat. 40400060-3, Bioworld^®). Optical density (OD) values were obtained by ELISA reader (Elisa reader, Model ELx 800, BioT, Winooski, VT, USA) at a wavelength of 405 nm.