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.