Serum samples and cells
A total of 342 serum samples were harvested from pigs infected with SVA. Antibodies directed against SVA in the serum samples were detected with the commercial BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit (South Lake Tahoe, State of California，CA,USA) and an indirect IFA. Serum samples and porcine kidney epithelial cells (IBRS-2) were obtained from the Key Laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China.
pSMK, pSMA and SUMO fusion protein expression vectors, were constructed as described previously (Xiao et al. 2016). The SUMO-tagging recombinant protein expression vectors containing the SVA VP0, VP1 and VP3 gene were constructed as described previously (Mo et al. 2019). In brief, the recombinant plasmids pSMA-VP0, pSMK-VP1 and pSMK-VP3 were constructed using pSMK and pSMA as the expression vector.
Expression of recombinant proteins
The recombinant plasmids were transferred into E.coliBL21(DE3). The E.coli cells were cultured in LB medium containing ampicillin (50 μg/ml) and kanamycin (10 μg/ml) at 37℃. When the OD600 was 0.7–0.9, Isopropylthio-β-d-galactoside (0.05 mM) was added to LB medium at 16℃ to induce coexpression of the recombinant proteins for 16 h.
Purification of recombinant proteins and quantification of VLPs
The His-SUMO-tagged recombinant protein was purified and analyzed with SDS-PAGE and western blotting, as previously described (Mo et al. 2019). The recombinant protein was incubated with SUMO protease at 4℃ to remove the His-SUMO tag, and perform VLP self-assembly in the same reaction system (Yin et al., 2010). A dynamic light scattering instrument (DLS) and transmission electron microscopy (TEM) were used to determine the molecular size and shape of the SVA VLPs, respectively (Guo et al., 2013).
Animal immunization and serum purification
Each of three adult rabbits was subcutaneously injected with VLPs (200 μg) and an equal volume of Freund’s complete adjuvant to induce antibody production against the antigen. Two booster immunizations with the same dose of VLPs plus Freund’s incomplete adjuvant were administered at 2-weekly intervals. Two weeks after the final booster injection, the blood of rabbits was collected, and the sera were prepared and stored at −80℃ before analysis.
Preparation of competitive antibody
The immunoglobulin G (IgG) in the rabbit sera obtained in the previous step was separated with the saturated ammonium sulfate method. The rabbit IgG was further purified with Protein A Sepharose affinity column chromatography and then labeled with horseradish peroxidase (HRP) with the improved NaIO4 method (Minaeian et al. 2012). The HRP-conjugated rabbit IgG was stored at –80℃ before use.
Establishment of cELISA method
A 96-well ELISA microplate was coated with various concentrations (0.5-1.0 μg/ml) of SVA VLPs in carbonate buffer solution (0.05 M, pH 9.6) and incubated at 4℃ overnight. After the microplate was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween (PBST), it was blocked with 1% bovine serum albumin (BSA) in distilled water for 1 h at 37℃, washed three times with PBST, and patted dry. In the antibody-coated wells of the plate, 50 μL of SVA-positive or SVA-negative serum and 50 μL of serially diluted HRP–IgG were mixed to ensure that the competitive reaction was as efficient as possible. The plates were incubated for 60 min at 37℃ and washed 3-4 times with 300 μL of PBST. Tetramethylbenzidine (TMB; 50 µl) was added and the samples incubated for 15 min at 37℃. The color reaction was stopped with 50 µL of 2 M H2SO4. To optimize the reaction conditions of the SVA cELISA, the best results under each set of condition were determined on the basis of the OD450 values and the percentage inhibition (PI), which was calculated with the following formula: PI = (OD450 of standard negative serum – OD450 of measured individual sample)/(OD450 of standard negative serum – OD450 of standard positive serum) × 100%.
Determination of the cELISA cut-off value (PI)
In this procedure, 50 SVA-positive seras with different antibody titers and 50 SVA-negative sera were tested by the established cELISA to determine the PI. The cut-off value was determined based on a receiver operating characteristic (ROC) curve.
Evaluation of the cELISA
Analysis of the sensitivity and specificity of the cELISA
The sensitivity of the cELISA was assessed with SVA-positive sera with different antibody titers. To determine whether the SVA cELISA system reacted positively with antibodies directed against viruses other than SVA, six randomly selected pig sera positive for serotype O FMDV, PCV2, porcine pseudorabies virus, Classical swine fever virus,Actinobacillus pleuropheumoniae, or Haemophilus parasuis were tested with the SVA cELISA.
cELISA repeatability test
A repeatability test was conducted to determine the stability of the SVA cELISA. The intra-assay repeatability of the SVA cELISA was tested with 10 randomly selected serum samples, with three replicates of each, in the same microplate. The inter-assay repeatability of the SVA cELISA was used to test the serum samples in triplicate in microplates in different production batches. The coefficients of variation (CVs) of the three replicates of each of the 10 serum samples were calculated.
Comparison of cELISA and commercial ELISA
To validate the SVA cELISA, a total of 342 swine sera samples were tested with both the SVA cELISA and the BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit.
Comparison of cELISA and IFA
When cells dense were about 60% in cell culture flasks, they were infected with the virus, and the virus solution was discarded at the appropriate time. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 10-15 min, and washed three times with PBS. The cells were treated with 0.1% Triton X-100 for 15 min and washed three times with PBS. An anti-SVA polyclonal antibody was added, and the cells were incubated at 37℃ for 2 h. The cells were washed three times with PBS, FITC-labeled anti-porcine IgG was added, and the cells were incubated for a further 1 h at 37℃ in an incubator. The cells were washed three times with PBS and observed under an inverted fluorescence microscope.