Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus

Figures

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  Figure 1. ASFV p30 antigen preparation. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of recombinant p30 protein, followed by Coomassie blue staining. (B) Western blot detection of His-tagged p30 protein. The membrane was stained with anti-His tag antibody. Molecular weight marker is shown on the left side. Arrows indicate purified p30 protein. (C) Antigen coating amount determined by testing a serially diluted p30 protein in indirect ELISA. x-axis represents the amounts of antigens, and y-axis shows the OD value.

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  Figure 2. Selection of p30-specific mAb for use in bELISA. (A) IFA performed on MARC-145 cells that were transfected with plasmid DNA expressing N-terminal or C-terminal fragment of p30. Cells were fixed and stained with a specific anti-p30 mAb (green) at 48 h post-transfection. Cell nucleus was counter-stained with DAPI (blue). Scale bars, 200 μm. (B) Positive and negative control sera were tested to determine the blocking effect for a biotinylated mAb. Each data point is shown as the mean value of two repeats, and the error bar represents the standard deviation. **** p < 0.0001; NS, statistically not significant.

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  Figure 3. Analytical sensitivity of p30-based bELISA. Two-fold serial dilutions of the high-positive and negative control serum were tested in bELISA. Each data point is shown as the mean value of two repeats, and the error bar represents the standard deviation. Asterisks indicate dilutions at which the PI and OD410 of the positive control serum were statistically different from those of negative control serum. **** p < 0.0001.

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  Figure 4. Determination of diagnostic sensitivity and specificity. Receiver operating characteristic (ROC) analysis was performed using 916 serum samples with known ASFV antibody status (810 negatives and 106 positives). (A) Interactive plot showing the cutoff value and optimal diagnostic sensitivity and specificity. (B) ROC curve showing the accuracy value interpreted as area under the curve (AUC). ROC analysis was performed using MedCalc, version 10.4.0.0 (MedCalc Software, Mariarke, Belgium).

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  Figure 5. Kinetics of antibody response in serum from ASFV-infected pigs. Serum samples were collected from six pigs infected by ASFV strain OURT 88/3 at 0, 3, 7, 10, 14 and 17 days post-inoculation. The dashed line represents cutoff of bELISA. x-axis shows different time points of sample collection. y-axis shows calculated percent inhibition. **** p < 0.0001; ** p < 0.01; ns, statistically not significant.