Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein

Figures

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  Figure 1. (a) SDS-PAGE of HEV-3 ORF2 110-610_6his protein. (b) Western blot with a monoclonal anti-HEV ORF2 ab. (c) Western blot with HEV-positive pig serum. M-pre-stained protein molecular marker (kDa), 1-total soluble protein extracted from plant leaves infiltrated with empty pEff. 2-total soluble protein extracted from plant leaves inoculated with pEff:HEV-3 ORF2 110-610_6his construct.

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  Figure 2. (a) SDS-PAGE of the purified HEV-3 ORF2 110-610_6his protein by immobilized metal-anion chromatography. (b) Western blot with an anti-HEV ORF2 mAb. M-presented protein molecular marker (kDa), 1-purified and dialyzed HEV-3 ORF2 110-610_6his protein, 2-protein extracted from non-inoculated plant leaves.

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  Figure 3. Analysis of the HEV-3 ORF2 110-610_6his protein. (a) Atomic force microscopy. (b) Transmission electron microscopy, scale bar = 200 nm. (c) Cryo-EM micrograph of hepatitis E virus particles. (d) TEM, scale bar = 20 nm; Arrows indicate VLPs.

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  Figure 4. Determination of optimal iEIA conditions. Five positive and five negative serum samples previously determined with commercial kit were used. (a) Optimal dilutions of positive serum samples. (b) Optimal dilutions of negative serum samples; Data are shown as absorbance values of the used serum samples; the bars refer to standard error. The presented absorbance values are average values with standard errors calculated from five different negative serum and five different positive serum, repeated in four independent experiments. (c) Optimal antigen concentration was determined. (d) Secondary antibody dilution was determined using an antigen concentration of 4 µg/mL, and serum dilution 1:50. P/N ratio in (c,d) was calculated at the relation A(λ492nm)/A(λ492nm) positive/negative control.

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  Figure 5. ROC analysis of the developed iEIA. ROC curve was generated using the results obtained by analyzing 72 negative and 69 positive pig serum samples, previously detected by PrioCHECK™ Porcine HEV Ab ELISA Kit. The red line shows the mean area under the curve (AUC) plot, with the AUC value of 0.9942 (95% Cl: 0.9672–0.999). NCSS 2021 Data Analysis software was used to perform the ROC analysis.