Development of a novel enzyme-linked immunosorbent assay to detect anti-IgG against swine hepatitis E virus

Abstract

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.

Keywords: ELISA; diagnosis; swine hepatitis E virus.

Fig. 1

Purification of the recombinant capsid protein of swine HEV. The recombinant capsid protein was analyzed by SDS-PAGE (A and C) and Western blotting with an anti-HEV monoclonal antibody (B). Western blotting with HEV-positive pig serum was also performed (D). Lane M: prestained protein molecular mass marker (kDa), lanes 1-4, and 6: induced capsid protein, lane 5: uninduced capsid protein.

Fig. 2

Determination of the optimal antigen concentration (recombinant capsid protein) for the developed ELISA. A two-fold dilution series of the recombinant capsid protein was performed to identify the optimum concentration of the recombinant protein, and a titration experiment was performed using pig sera already known to be HEV-positive. Results of the checkerboard titration analysis demonstrated that the most optimal and reliable results were obtained when each microplate well was coated with 1.25 µg/mL of the capsid protein.

Fig. 3

Receiver operating characteristics (ROC) analysis of the developed ELISA. The area under the ROC curve (AUC) of the developed ELISA was 0.987 (95% CI, 0.977~0.998; p < 0.001). The blue line represents the test curve and the green line corresponds to the non-informative test curve. Sensitivity and specificity of the developed ELISA were 97% and 95%, respectively, when the optimal cut-off OD value was 0.6.

Fig. 4

Correlation between the results of the developed ELISA and a commercial kit for 235 field-collected pig sera samples. After the 235 pig sera were tested with the HEV-specific commercial ELISA kit, the same 235 samples were subjected analysis with the developed ELISA test. The correlation ratio (R²) between the developed ELISA and the commercial kit results was 0.717.