I329L protein-based indirect ELISA for detecting antibodies specific to African swine fever virus

Figures

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  Figure 1. Identification of the recombinant ASFV I329L protein. (A) SDS-PAGE analysis of the recombinant ASFV I329L protein. M: protein marker; 1: protein from uninduced transformed (E) coli; 2: protein from induced transformed (E) coli; 3: purified protein. (B) Identification of the recombinant ASFV I329L protein using an ASFV-positive serum via western blot. M: protein marker; 1: purified protein.

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  Figure 2. Antigenicity of the recombinant truncated ASFV I329L protein. The full-length I329L recombinant plasmid pCAGGS-EGFP-fI329L was transfected into HEK-293T cells and then incubated with the antiserum (1:500 dilution) of the mouse inoculated with the truncated I329L protein. Red fluorescence showed that the mouse antiserum reacted specifically with the I329L eukaryotic expression protein in 293T cells. (A) 293T cells were only stained with DAPI and showed blue fluorescence; (B) 293T cells were transfected with the pCAGGS-EGFP-fI329L plasmid and showed green fluorescence; (C) The 293T cells transfected with recombinant plasmid were incubated with the positive mouse sera and the goat anti-mouse secondary antibody in order, and observed with red fluorescence; (D) The former three images were merged.

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  Figure 3. Optimization of the developed ASFV I329L-based iELISA. The optimal working concentration of the coating antigen and pig sera was determined using checkerboard titrations. The ratios of positive to negative reference serum (P/N) are presented in the heatmap. Darker colors indicate higher P/N OD450 ratios.

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  Figure 4. ROC curve of the determination of the iELISA cut-off value. ROC curve analysis was performed using SPSS software using the OD450 values from thirty-five positive inactivated sera and sixty negative sera detected by the iELISA.