Optimization of S-iELISA
To develop an indirect ELISA for detecting SADS-CoV IgG antibodies, a checkerboard titration was performed to determine the optimal work conditions of S-iELISA based on the standards that the OD450nm value of positive serum was close to 1.0, the P/N value was highest, and the negative serum was low. As shown in Fig 3, the optimal dilution of coated antigen S-Fc protein and second antibody dilution were set at 4 μg/well and 1:8000. Furthermore, the optimal serum sample dilution was defined as 1:200. After the above-mentioned conditions were determined, it was found that 5% skimmed milk in 1 × PBST was the best blocking solution and the best blocking time for 2 h at 37°C were sufficient and time-saving in the S-iELISA. In addition, the optimal reaction times for serum samples and second antibody were 2 h and 45 min, respectively. Finally, the optimal stopping time was determined as 20 min.
Cut-off value, repeatability and specificity of S-iELISA
To determine the cut-off value of the S-iELISA, 40 SADS-CoV-seronegative samples, verified by both IFA and Western Blot assays, were tested by this ELISA method. As shown in Fig 4A, the mean OD450nmvalue of these negative serum samples was 0.2870, and the SD of these samples was 0.0326. Therefore, the cut-off value of S-iELISA was calculated to be 0.3711, indicating that the sample OD450nm value≥0.3711 was considered to be SADS-CoV-seropositive and vice versa. In the repeat ability experiment, six SADS-CoV-positive serum samples and one SADS-CoV-negative serum sample were used to determine the intra- and inter-assay CV of the S-iELISA, which was 0.49%-2.09% and 2.41%-5.20%, respectively (Fig 4B). To determine the specificity of S-iELISA, porcine positive sera against common swine viruses were examined. As shown in Fig 4C, the average OD450nm values of positive serum samples for PEDV, TGEV, PDCoV, FMDV, ASFV, CSFV, PRV, PRRSV and PCV-2 were 0.2507, 0.3018, 0.282, 0.2732, 0.1881, 0.2140, 0.2659, 0.2205, 0.2686, respectively. These values are all less than 0.3711, indicating that these serum samples were PEDV-seronegative and non-cross-reactive with this S-iELISA.