Development of an Indirect ELISA to Distinguish between Porcine Sapelovirus-Infected and -Vaccinated Animals Using the Viral Nonstructural Protein 3AB

Figures

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  Figure 1. Purification and characterization of PSV 3AB. (A) Lane 1 is the pET-28a (+) control, lane 2 is the pET-28a-3AB after induction, lane 3 is the supernatant of the pET-28a-3AB after induction, and lane 4 is the inclusion bodies of the pET-28a-3AB after induction. (B) Lane 1 represents the flow-through liquid, lane 2 represents the washing process using washing buffer, and lanes 3–6 represent the 500 μL fractions collected sequentially using buffer containing 200 mM imidazole. (C) Western blot analysis of the recombinant 3AB protein. M is the protein ladder. The arrow indicates the location of the 3AB protein.

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  Figure 2. Optimization of the ELISA reaction conditions. (A) Screening of coating antigen concentration and serum dilution. (B) Screening of the blocking solution concentration. (C) Screening of the HRP-IgG dilution and incubation time. (D) Screening of the TMB reaction time.

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  Figure 3. Diagnostic performance of the indoor ELISA was evaluated using serum from healthy, vaccinated, and infected animals. (A) ROC curve analysis was performed on the established 3AB ELISA with healthy (n = 41), vaccinated (n = 60), and infected (n = 50) serum. Healthy and vaccinated samples were used as uninfected controls. (B) According to the sensitivity, specificity, and 95% confidence interval (CI), the best threshold was determined by dot plot analysis.

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  Figure 4. The diagnostic performance of the indoor ELISA was evaluated using serum from healthy, vaccinated, and infected animals. (A) ROC curve analysis was performed on the established 3AB ELISA with healthy (n = 41), vaccinated (n = 60), and infected (n = 50) serum. Healthy and vaccinated samples were used as uninfected controls. (B) According to the sensitivity, specificity, and 95% confidence interval (CI), the best threshold was determined by dot plot analysis.