Double-antigen sandwich ELISA based on S1 for cross-species detection of Porcine deltacoronavirus antibodies

Fig. 1. Expression, purification, and validation of the PDCoV S1 protein. A SDS-PAGE (10%, reducing) analysis of Ni–NTA-purified S1 protein from cell culture supernatants. Molecular-mass markers (kDa) are indicated on the left. B Western blot probed with PDCoV-positive pig serum (1:200) followed by HRP-conjugated goat anti-pig IgG (1:10,000; Abcam, Cambridge, UK). C Indirect ELISA verification using the purified S1 protein (100 ng/well) as the coating antigen. Sera from PDCoV-positive (n = 4) and negative (n = 4) pigs were tested at a 1:20 dilution, followed by detection with an HRP-conjugated goat anti-pig IgG (1:2,000)

Fig. 2. Optimization of DAgS-ELISA parameters. A Coating antigen concentration and HRP-labeled antigen dilution determined by checkerboard titration. B Optimization of the dilution ratio of the test serum. C Optimization of coating time and temperature. D Comparison of blocking buffers. E Optimization of incubation time and temperature. F Optimization of serum incubation time.. G Optimization of the detection antigen reaction time. H Substrate development time. PDCoV-positive and -negative sera served as controls, and all parameters were evaluated using the positive-to-negative (P/N) ratio

Fig. 3. Determination of the cut-off value and diagnostic performance of the DAgS-ELISA. A Receiver operating characteristic (ROC) curve analysis showing the assay’s discrimination between PDCoV-positive and PDCoV-negative sera, with an area under the curve (AUC) of 0.995. (B) Optimal OD₄₅₀ cut-off value (0.16) determined from the ROC analysis, corresponding to a diagnostic sensitivity of 92.86% and specificity of 99.11%

Fig. 4. Analytical evaluation of the DAgS-ELISA. A Detection limit. Serial two-fold dilutions of a PDCoV-positive serum were tested. The assay detected antibodies at a dilution of up to 1:2048, demonstrating high sensitivity. B Cross-reactivity. Testing the assay against positive sera for common swine pathogens (PEDV, PRV, PoRV, FMDV, CSFV, PCV2, PRRSV, TGEV) showed no signal above the cut-off (dashed line), demonstrating no cross-reactivity

Fig. 5. Cross-species detection of PDCoV antibodies using the DAgS-ELISA. PDCoV-positive and -negative sera from pigs, rabbits, mice, and chickens were tested. Each serum sample was analyzed in triplicate (n = 3). The assay results demonstrate the ability of the DAgS-ELISA to detect PDCoV antibodies across multiple species

Fig. 6. Correlation between the DAgS-ELISA and the virus neutralization test (VNT). A Distribution of individual values obtained by both assays. B Pearson correlation between DAgS-ELISA optical density values and VNT titers; p < 0.05 was considered statistically significant