Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV

Figures

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  Figure 1. SDS-PAGE and western blot analysis of purified recombinant CD2v (63 kDa) and p30 (35 kDa) proteins (A). Analysis of immunogenic identification of p30 and CD2v proteins by western blot assay (B–D). Recombinant CD2v and p30 proteins could react with ASFV-positive sera (B), but not-negative sera (C). The recombinant p30 protein could react with ASFV-ΔCD2v-infected sera, in contrast to the CD2v protein (D). Lane 1: protein marker; Lane 2: CD2v; Lane 3: p30.

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  Figure 2. Optimization of ELISA conditions for antibody detection of ASFV CD2v or p30. P/N ratios of an ASFV-positive and -negative serum using different concentrations of CD2v and p30 (A). P/N ratios of an ASFV-positive and -negative serum using HRP-labeled secondary antibody of different concentrations for CD2v and p30 (B). P/N ratios of an ASFV-positive and -negative serum with various dilutions of serum samples and various reaction times of the HRP-labeled secondary antibody (C,D). P/N ratios of an ASFV-positive and -negative serum with various reaction times of serum samples and TMB substrate solution, respectively (E,F). Data are shown as the mean ± SD. Blue lines with squares indicate CD2v, whereas red lines with triangles indicate p30.

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  Figure 3. Determination of the cut-off values for CD2v and p30 ELISA. A total of 43 serum samples were detected using the OIE-recommended INgezim ASFV antibody blocking ELISA kit (A). These serum samples were also detected using the newly established ELISA for CD2v and p30 proteins (B).

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  Figure 4. Detection sensitivity and specificity of the ELISA kits based on the CD2v or p30 proteins for ASFV. Ten ASFV-infected serum samples were subject to a series of dilutions. According to the cut-off value, the positivity of these serum samples could be detected up to a dilution of 5120 times (A). The two ELISA kits based on CD2v or p30 proteins did not react with 1:40 diluent CSFV-, PRRSV-, JEV-, PRV-, and PPV-infected serum samples, highlighting their specificity (B). Data are shown as the mean ± SD. Blue lines with squares indicate CD2v, whereas red lines with triangles indicate p30.

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  Figure 5. The dual ELISA was used for the detection of ASFV- or ASFV-ΔCD2v-infected serum samples. The INgezim ASFV antibody blocking ELISA kit were used to detect ASFV- and ASFV-ΔCD2v-infected and negative serum samples (A). The newly established ELISA based on p30 were used to detect ASFV- and ASFV-ΔCD2v-infected and negative serum samples (B). The newly established ELISA based on CD2v were used to detect ASFV- and ASFV-ΔCD2v-infected and negative serum samples (C). The ELISA based on medium from CHO cells transfected with empty plasmid were used to detect ASFV- and ASFV-ΔCD2v-infected and negative serum samples (D).