Serum samples
Serum samples from naïve animals: Serum samples from clinically healthy and unvaccinated pigs (n = 102) were collected and tested using Swinecheck® SVA bELISA kits (all samples had negative results, blocking rate < 50%). These samples were used to assess diagnostic specificity (Dp) and the cutoff value.
Serum samples from vaccinated animals: A total of 124 serum samples were collected at 28–42 days post-vaccination (dpv) with an inactivated whole-virus vaccine and collected by our research group. These samples were used to assess diagnostic sensitivity (Dn) and the cutoff value.
Serum samples from infected animals: A total of 23 serum samples from swine infected with SVA ZJ/2015 at 7–14 days post-infection (dpi) were collected by our research group. These serum samples were used for epitope mapping after heat inactivation.
Field samples (partial samples seropositive for other vesicular diseases): A total of 167 serum samples from swine were collected in the field and preserved at -80°C. These serum samples were used to compare accuracy rates and diagnostic performances.
Standard control sera: SVA-positive sera (blocking rate 101.2%, measured by Swinecheck® SVA bELISA kit and confirmed by VNT) were collected from the vaccinated group, and SVA-negative sera (blocking rate 9.2%, measured by Swinecheck® SVA bELISA kit and confirmed by VNT) were collected from the naïve group. A standard positive control (P) and negative control (N) were created as internal controls.
Growth and purification of SVA
NCI-H1299 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. The SVA ZJ-2015 strain was preserved in our laboratory. Briefly, NCI-H1299 cells infected with SVA ZJ-2015 were collected and subjected to three freeze‒thaw cycles. Cellular debris was removed by centrifugation at 6000 × g for 30 min at 4 °C. The virus was then precipitated with 8% polyethylene glycol (PEG 8000) and 0.5 M NaCl for 16 h at 4 °C. The precipitated virus was further centrifuged by a 10–50% sucrose density gradient. Subsequently, the virus band was taken up for desucrose treatment, and the precipitate was resuspended in PBS (pH = 7.4) and stored at − 80°C. Purified virus was used for the preparation of infectious and immune sera.
Neutralization assay
The neutralizing activity of sera was determined by end-point dilution assay. The test sera were heat-inactivated at 56 °C for 30 min and then twofold serially diluted in DMEM (50 µl/well). Each dilution was repeated in triplicate. An equal volume of 100 median tissue culture infective dose (TCID50) SVA ZJ-2015 was added to each well of a 96-well tissue culture microtiter plate (50 µl/well). The plates were incubated for 1 hr at 37 °C. Then, 100 µl of NCI-H1299 cells (2×104 cells) in DMEM were added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. The cytopathic effect (CPE) was scored after 72 hr. The neutralizing antibody titer was calculated by the Reed–Muench method. In brief, a 100% CPE on cells in the tested serum wells was judged as negative, and the presence of more than 50% of cells remaining was judged as positive. Finally, the serum dilution that protected 50% of the cell wells from cytopathy was calculated, and this dilution was the serum neutralizing antibody titer.
Epitope mapping of VP2 protein
To identify the epitope recognized by SVA-infected pig serum, overlapping peptides of the VP2 protein (15 amino acids in length, overlapping each other by 10 amino acids) were synthesized by GenScript Biotech Corporation (Nanjing, China). The synthesized peptides were then screened and identified by indirect ELISA.
Construction and optimization of VP2-epitp-ELISA
ELISA plates (Costar, catalog number: 42592) were coated at 4 °C overnight with 100 µl of synthesized peptides (200 ng/well, 100 ng/well, 50 ng/well, 25 ng/well) diluted in PBS buffer (pH 7.4). The plate was then thoroughly washed with PBST (PBS containing 0.05% Tween-20) and blocked with PBST containing 1% BSA and 5% sucrose at 37 °C for 2 h. After five PBST washes, positive and negative control serum samples were diluted (1:10, 1:20, 1:40, 1:80) in the abovementioned sample diluent, diluted sera were transferred to coated plates at 100 µl per well, and the plate was incubated at 37 °C for 30 min. Then, after five washes, each well received 100 µl of 1:10000 diluted secondary antibodies and rabbit anti-pig IgG antibodies conjugated to horseradish peroxidase (Sigma, USA) for 30 min at 37 °C. After washing, the plate was developed with 100 µl of TMB substrate at 37 °C for 10 min, and the reaction was terminated with 100 µl of 2 M H2SO4. The absorbance at 450 nm (A450) was measured using a Varioskan Lux instrument.
Estimation of the cutoff value, Dn and Dp for VP2-epitp-ELISA
After the optimum coating concentration and serum dilution were confirmed, pig serum samples with a known status were tested using VP2-epitp-ELISA to evaluate their Dp, Dn and cutoff value. The A450 values of the positive control (A450 pos) and test samples (A450 sample) were corrected by deducting the A450 value of the negative control (A450 neg). The sample results were recorded as percent positive (PP) using the following formula: PP = (A450 sample – A450 neg) ×100%/ (A450 pos – A450 neg). The A450 values were also recorded as the percent inhibition (PI) using the same formula. All PP values of the assay were used to estimate the cutoff value, Dp and Dn using MedCalc software.
Comparison of accuracy rates and diagnostic performances
To evaluate the accuracy rates and concordance rate of VP2-epitp-ELISA, a total of 249 serum samples (serum samples from naïve animals: 102; serum samples from vaccinated animals: 124) and a total of 167 field samples (partial samples seropositive for other vesicular diseases) were tested using Swinecheck® SVA bELISA.