Development of a Nucleocapsid Protein-Based Blocking ELISA for the Detection of Porcine Deltacoronavirus Antibodies

Figure 1. Identification of the recombinant PDCoV N protein. (A) SDS-PAGE analysis of the re-combinant PDCoV N protein. (B) Western blot identification of the recombinant PDCoV N protein using a mouse anti-His monoclonal antibody (mAb). (C) Western blot identification of the recom-binant PDCoV N protein using the mAb 1A3. Lane 1, Prestained Protein Ladder; Lane 2, super-natants of the bacterial lysates; Lane 3, precipitates of the bacterial lysates; Lane 4, the purified PDCoV N protein.

Figure 2. Optimization of the developed PDCoV N-based blocking ELISA. (A) Determination of the optimal working concentration of coating antigen and mAb 1A3 by checkerboard titrations. Theoptical density at 450 nm (OD450nm); ratios of positive to negative reference serum (P/N) are presented in a heatmap, which was drawn with the GraphPad Prism software. The darker the color, the greater the P/N OD450nm ratio. (B) Comparison of the blocking effect of three blocking buffers (2% bovine serum albumin (BSA), 5% skimmed milk and 0.4% gelatin). (C) Determination of the optimal working dilution of the horseradish peroxidase (HRP)-conjugated Goat anti-Pig IgG secondary antibody. (D) Determination of the optimal immunoreaction time for both serum samples and HRP-conjugated secondary antibody. All results were presented as the mean ± SD of triplicate experiments. The black circles represent the P/N values of three independent runs.

Figure 3. Cut-off value determination, diagnostic sensitivity and specificity analysis of the PDCoV N protein-based blocking ELISA. Receiver operating characteristic (ROC) analysis was performed using 384 swine serum samples with known PDCoV antibody statuses, including 330 positives and 54 negatives. (A) ROC curve showing the accuracy value was reflected by the area under the curve (AUC). ROC analysis was performed using GraphPad Prism software (Version 5.0; La Jolla, CA, USA). (B) Interactive plot showing the optimal percent inhibition (PI) cut-off value and diagnostic sensitivity and specificity. The optimal cut-off value for the blocking ELISA was calculated using the Youden index.

Figure 4. IFA analysis of the mAb 1A3 raised against the PDCoV N protein. LLC-PK1, ST, MA104, and Vero cells were mock-infected or infected with PDCoV, TGEV, PRoV, and PEDV or PEAV, respectively, at a multiplicity of infection of 1 for 48 h post-infection. The cells were fixed and processed for immunostaining with monoclonal antibody1A3 or PDCoV antiserum, followed by staining with fluorescein-isothiocyanate-conjugated Goat anti-Mouse IgG or Rabbit anti-Pig IgG. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Scale bars, 200 µm.

Figure 5. Analytic specificity of the developed PDCoV N-based blocking ELISA. Antisera against PDCoV, PEDV, TGEV, PRoV and PEAV were tested via the ELISA. The PI cut-off value of 51.65% was marked with a dashed line. The black circles represent the PI values of three independent runs on each antiserum.

Figure 6. Analytic sensitivity of the developed PDCoV N-based blocking ELISA. Two-fold serially diluted positive reference PDCoV antiserum ranging from 1:8 to 1:2048 were detected via the ELISA. The PI cut-off value of 51.65% was marked with a dashed line. The black circles represent the PI values of eight independent runs on each dilution.