SADS-CoV S protein indirect ELISA (S-iELISA) development
Conventional indirect ELISA detection serum antibodies as described
previously with some modifications (Lin et
al., 2018). ELISA plates with 96 wells (Costar, USA) were coated with
100 μL/well of purified recombinant S-Fc protein in 50 mM bicarbonate
buffer (pH = 9.6) and incubated overnight at 4 °C. After three washs
with PBST (1 × PBS containing 0.05% Tween-20), the plates were blocked
with 5% NON-Fat Powdered Milk (Shanghai Sangon Company, China) in 1 ×
PBST for 2 h at 37 °C. After plates were washed three times, 100 μL/well
porcine serum samples diluted in 1 × PBST was added and incubated for 1
h at 37 °C. After the same washing step, plates were reacted with 100 μL
diluted secondary antibody
(HRP-goat
anti-pig IgG, Solarbio, China) for 30 min at 37 °C for the purpose of
detecting IgG against SADS-CoV in serum samples. Plates were then washed
three times and the peroxidase reaction was visualized using 100 μL
3,3′,5,5′-tetramethylbenzidine (TMB) (KPL, USA) as the substrate for 15
min at 37 °C in complete darkness and stopped by adding 50 μL of 2 M
H2SO4 to each well. Finally, the
OD450nm values was measured and recorded immediately
using
Spectra-Max
M2 (Molecular Devices, USA). Positive, negative and blank (1 × PBST)
samples were tested in duplicate and included on each plate.
The optimal concentration of coating antigen and second antibody
dilution for the S-iELISA were determined using
a
checkerboard titration as described previously with some modifications
(Li et al., 2015). Briefly, the
concentration of S-Fc protein was coated on 96-well microtiter plates
gradually reduced from 0.25 μg/mL to 8 μg/mL. The standard pig
SADS-CoV-positive serum and negative serum were diluted in 1:100. After
the antigen and antiserum were added, HRP-labeled secondary antibody was
added correspondingly to the plate with dilutions from 1:1000 to 1:8000
to determine the optimal conjugate dilution. When the conditions that
gave the highest OD450nm ratio between the positive and
negative serum (P/N value) and an OD450nm value for
positive serum close to 1.0 with the OD450nm value of
negative serum ≤ 0.2 were consider optimal.
After the conditions mentioned above were determined, the serum
were
diluted in serial twofold dilutions from 1:100 to 1:3200. Then, the
blocking buffers with 1% BSA, 5% skimmed milk, 5% fetal bovine serum,
10% goat serum were used and blocked for 1, 2, 3 and 4 h at 37°C. The
incubation time of serum sample was optimized with 0.5, 1, 1.5, 2 h. The
HRP-goat anti-pig IgG was optimized with 15, 30, 45, 60 min. The
reactions were stopped and optimized by assessing 10, 15, 20, and 25
min, respectively.