Development and Comparison of Indirect ELISAs for Detecting IgG and IgA Antibodies Against Major Structural Proteins of Porcine Deltacoronavirus With Virus Neutralization as a Benchmark

Figures

• 

  Figure 1. Expression, purification, and reactivity analysis of recombinant PDCoV proteins. (A) SDS-PAGE analysis of purified recombinant PDCoV proteins. Lane M: Protein molecular weight marker; lane 1: S protein (~180 kDa); lane 2: S1 protein (~70 kDa); lane 3: RBD protein (~20 kDa); lane 4: M protein (~65 kDa); lane 5: N protein (~42 kDa). (B) Western blot analysis of the purified proteins using an anti-His tag antibody. Lanes are labeled as in (A). (C–D) Assessment of PDCoV antigen immunoreactivity. Red and blue bars: mean gray values of five PDCoV-positive sera; gray bars: mean values of three negative-control sera; line graph: corresponding P/N ratios.

• 

  Figure 2. Determination of cut-off values and diagnostic performance of the iELISAs. For each antigen-based assay (A–G), the left panel presents the ROC curve plotting sensitivity against 1-specificity, with the AUC indicating overall accuracy. The right panel displays a dot plot of the S/P ratios obtained from testing known PDCoV-positive and negative serum samples. The horizontal dashed line in each dot plot indicates the optimal cut-off value (determined by maximizing Youden's index).

• 

  Figure 3. Analytical specificity and sensitivity of the PDCoV iELISAs. (A) PEDV-specific antibody levels in serum, determined by commercial ELISA kits. (B) Neutralizing activity against PDCoV, as determined by VNT. (C, D) Cross-reactivity of the IgG (C) and IgA (D) iELISAs (S-, S1-, RBD-, and N-based) with PEDV-positive serum (n = 20). (E, F) Reactivity of the IgG (E) and IgA (F) iELISAs with antisera against other porcine pathogens (PCV2, PCV3, PRRSV, PRV, PoRV, CSFV, ASFV). (G, H) Analytical sensitivity of the IgG (G) and IgA (H) iELISAs, determined by testing two-fold serial dilutions (1:2 to 1:128) of a PDCoV-positive antiserum. The horizontal dashed line in panels (A) and (C–H) indicates the assay-specific cut-off value (determined by Youden's index).

• 

  Figure 4. Antibody kinetics in vaccinated pigs and diagnostic sensitivity of iELISAs. (A) Schematic of the animal immunization and sample collection protocol. (B) Neutralizing antibody titers in serum, as determined by VNT. (C–F) Dynamics of IgG antibodies against S, S1, RBD, and N proteins, measured by iELISA. (G,H) Dynamics of IgA antibodies against S1, and RBD proteins, measured by iELISA. The horizontal dashed line in panels (C–H) indicates the assay-specific cut-off value.

• 

  Figure 5. Correlation of iELISA S/P ratios with neutralizing antibody titers. Pearson correlation analysis between S/P ratios (from S1- and RBD-based IgG/IgA iELISAs) and NTs in clinical serum (A–D) and colostrum (E–H) samples. The correlation coefficient (r) and statistical significance (p-value) are shown for each analysis.