2.2 Serological testing of N-DRV
N-DRV antibody levels in the serum were detected by indirect enzyme-linked immunosorbent assay (ELISA). Briefly, the purified σC protein (50 ng/μL, prepared in our previous study ) was added into each well of the ELISA plates (NEST Biotechnology, Wuxi, China) and incubated for 12 h at 4℃, and then the antigen coating solution in the ELISA plates were completely aspirated, and plates were washed three times with washing buffer ( PBS containing 0.05% Tween-20, PBST ). Add 200μL of 5% skim milk powder (Solarbio, Beijing, China; w/v) to each well for blocking, and incubate at 37℃ for 2 h. After blocking, discard the blocking solution and wash 3 times. The duck serum sample (1:10) to be tested was added to the wells. A negative control and a blank control group were set. After incubating at 37℃ for 1 h, the samples were washed three times with PBS and the washing solution in the wells was blotted. Then, the plates were incubated with rabbit anti-duck immunoglobulin G (IgG) at 1:1000, incubated again for 1h at 37℃, and were washed three times with PBS. After which, 100μL 3’3’5’5’-tetramethylbenzidine (TMB) substrate solution (TransGen, Beijing, China) was added to each plate and left to develop at 37 ° C in the dark for 15 minutes. So far, stop buffer (3M H2SO4) was add to stop the reaction at 100 μL /well (S. Zhang et al., 2020). The optical density (OD) values at 450 nm of each sample was measured using an ELISA microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The cutoff value was an OD of 0.459 at 450 nm. Each sample is repeatedly tested 3 times to ensure the accuracy of the test results.