All reagents for isolating viral RNA and PCR amplification were purchased from Invitrogen (USA). Pig sera were obtained from jugular vein of pigs in Korean pig farms and isolated with centrifugation at 750 × g for 10 min. HEV viral RNA was purified from pig sera using Trizol LS reagent (Invitrogen) according to the manufacturer's instructions. The viral RNA was eluted with a total volume of 30 µL RNase-free water and stored at -70℃ until further analysis. cDNA was synthesized using an external reverse primer specific for the capsid gene of HEV and M-MLV (moloney murine leukemia virus) Reverse Transcriptase. Briefly, 10 µL of viral RNA and 1 µL of external reverse primer (2 pmol/µL) were mixed, incubated at 70℃ for 10 min, and chilled at 4℃ for 5 min. This mixture was added to 4 µL of 5× First Strand Buffer that contained 2 µL of 0.1 M DTT (dithiothreitol), 1 µL of 10 mM dNTP mixture, 1 µL of RNase free water, 0.5 µL of RNaseOUT RNA inhibitor, and 0.5 µL of M-MLV Reverse Transcriptase. The samples were incubated at 37℃ for 1.5 h, 70℃ for 10 min, and 4℃ for 5 min before being stored at -20℃.

Two-step PCR amplification was performed using cDNA made from HEV isolated from swine serum as previously described [2]. The nested forward primer sequence was slightly modified for cloning: 5'-CACCAACCCTCT CTTGCCTCT-3'. The 724 bp PCR product corresponding to the capsid gene was cloned into the Champion pET-100/TOPO vector (Invitrogen, USA) in TOP10 competent Escherichia (E.) coli cells (Invitrogen) according to the manufacturer's protocol. The sequence of the resulting construct was confirmed with automatic-dye-terminator DNA sequencing (ABI Prism 377 L; Applied Biosystems, USA). The cloned plasmid was used to transform BL21 Star E. coli cells (Invitrogen) for expression.

Bacteria containing the cloned capsid gene were grown by adding 100 mL of seed culture to 1 L of LB broth containing 100 µg/mL ampicillin and cultured at 37℃ for 1.5 h with shaking at 200 rpm. Next, 1 mM isopropyl-β-D-thiogalactoside (IPTG; Duchefa Biochemie, The Netherlands) was added and culturing was continued for 5.5 h with periodic mixing. The cells were harvested by centrifugation at 750 × g for 20 min at 4℃, and resuspended in 40 mL of 10 mM imidazole lysis buffer (20 mM Tris, 500 mM NaCl, 8 M urea, 10 mM imidazole, and 1 mM β-mercaptoethanol, pH 8.0 in distilled water). The cells were lysed with a repeated freeze-thaw process. The lysate was purified using an His-spin Trap (GE Healthcare, UK) with the 10 mM imidazole lysis buffer and a 500 mM imidazole elution buffer (20 mM Tris, 500 mM NaCl, 8 M urea, 500 mM imidazole, and 1 mM β-mercaptoethanol, pH 8.0 in distilled water). The concentration of the purified recombinant capsid protein (6.3 mg/mL) was measured with a BCA Protein Assay Kit (Pierce, USA).

The HEV capsid protein was expressed after cloning the ORF2 gene (481~1,200 bp) into a pQE-30 UA vector (Invitrogen) as previously described [2]. The expressed protein was purified and used to immunize a BALB/c mouse by injecting the protein with Freund's incomplete adjuvant twice at a 2-week interval. Next, the inguinal lymph node was isolated and fused with SP2/0 Ag14 myeloma cells to produce monoclonal antibody. The hybridoma clone producing a monoclonal antibody specific for HEV was selected by performing an ELISA. 3F9 cell clone (IgG2b) was selected and used for the present study.

The purified recombinant capsid protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. Western blot analysis was performed by electrotransferring the separated proteins from the SDS-PAGE gel onto an iBlot Gel Transfer Stack nitrocellulose membrane (Invitrogen). The membrane was incubated with mouse anti-swine HEV monoclonal antibodies at a 1 : 5,000 dilution and an alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (H+L; Bethyl Laboratories, USA) at a 1 : 2,000 dilution. Antibody binding was detected using an AP conjugate substrate kit (Bio-Rad, USA). The purified recombinant protein was immunoblotted with pig serum that had been determined to be HEV-positive with a commercial ELISA kit (Diagnostics; Genelabs Technologies, Singapore) at a 1 : 1,000 dilution and an alkaline phosphatase-conjugated rabbit anti-pig IgG (H+L; Bethyl Laboratories) at a 1 : 2,000 dilution. The blots were developed with the AP conjugate substrate kit.

Optimal working dilutions of the recombinant capsid protein, pig serum, and horseradish peroxidase (HRP) conjugate were determined by checkerboard titration. The optimal concentration of the purified protein was verified using pig sera known to be positive or negative for swine HEV. Briefly, wells in the first lane of the ELISA plates were coated with 10 µg/mL of the recombinant protein in PBS and serially diluted two-fold. Aliquots of pig sera diluted two-fold (from 1 : 100 to 1 : 3,200) in antibody diluent [PBS containing 0.1% Triton X-100 (PBST) and 5% horse serum] were dispensed into the wells of the plates. HRP-conjugated goat anti-pig IgG (Bethyl Laboratories) in antibody diluent was added at a dilution of 1 : 1,000 or 1 : 2,000.

A total of 235 field pig sera samples collected in Korea were tested using a commercial HEV ELISA kit (Diagnostics; Genelabs Technologies) and HRP-conjugated rabbit anti-pig IgG (Sigma-Aldrich, USA) according to the manufacturer's instructions. The same sera were also analyzed with the ELISA developed in the present study using the recombinant protein. Briefly, 96-well ELISA plates were coated with 250 ng/well of the purified HEV capsid protein in PBS and incubated overnight at 4℃. The ELISA plate was then washed three times with 250 µL of PBST and blocked with 250 µL of PBS containing 10% horse serum at 37℃ for 1 h. After washing three times, 100 µL of pig sera diluted 1 : 100 in antibody diluent were added to each well and the plate was incubated at 37℃ for 1 h. The plates were again washed three times with PBST, and then 100 µL of HRP-conjugated goat anti-pig IgG (Bethyl Laboratories) diluted 1 : 2,000 in PBST was added. The plates were subsequently incubated for 1 h at 37℃. After washing three times with PBST, 100 µL of an HRP substrate solution was added to each well and the plate was incubated for 15 min at room temperature. Absorbance was read at an adjusted optical density (OD) of 405 nm using an Emax Precision Microplate Reader (MDS Analytical Technologies, USA). The cut-off value was determined as previously reported using negative pig sera [10]. All samples were assayed in triplicate.

The sensitivity, specificity, efficiency, and Youden index values were calculated to evaluate the diagnostic accuracy of the novel ELISA. The following formulae were used to calculate the ELISA sensitivity, specificity, efficiency, and Youden index values: Sensitivity = no. of true positive/(no. of true positive + no. of false negative); Specificity = no. of true negative/(no. of true negative + no. of false positive); Efficiency (%) = [(no. of true positive + no. of true negative)/(no. of true positive + no. of false positive + no. of true negative + no. of false negative)] × 100; Youden index = sensitivity + specificity - 1. The Youden index measures the probability of correct classification that is invariant to prevalence [6]. Values for the area under the receiving operator characteristic (ROC) curve of the developed ELISA were evaluated using the Statistical Package for the Social Sciences, 12.0 (SPSS, USA) at a 95% confidence interval (CI) [9,12].

The identity of the cloned gene was confirmed as the swine HEV capsid gene by sequencing of the gene. The swine HEV isolate belonged to genotype 3 and the sequence (GenBank accession No. 01-18934D2, AF466681; 01-19248-3, AF466660) was the same. The capsid protein was purified via elution with an imidazole gradient, and yielded a purified capsid protein concentration of 6.3 mg/mL (maximum). SDS-PAGE analysis demonstrated that the recombinant capsid protein had an approximate molecular mass of 35 kDa, which was slightly higher compared to the software-predicted molecular weights (Fig. 1A). However, Western blot analysis using the anti-HEV monoclonal antibody revealed the shifted band to be the recombinant capsid protein with monoclonal antibody (Fig. 1B and C) and known positive pig serum (Fig. 1D).

Concentrations of the recombinant capsid protein along with dilution ratios of the field-collected pig sera and conjugate were optimized using a checkerboard titration assay with pig sera confirmed to be HEV-positive or -negative with a commercial ELISA kit. The optimal concentration of recombinant capsid protein used to coat the ELISA plate was 1.25 µg/mL using a 1 : 100 dilution. This produced the highest positive/negative ratio for the standard checkerboard titration. The largest differences between absorbance of the positive and negative control sera samples were obtained by the addition of a 1 : 2,000 dilution of the conjugate to wells coated with the recombinant capsid protein under the optimized conditions (Fig. 2).

The 235 pig sera samples collected in the field were analyzed with a commercial HEV-specific ELISA kit. Results of this analysis were compared to those from the ELISA developed in this study using the recombinant capsid protein. The ROC analysis revealed that the area under the curve for the developed ELISA was 0.987 (95% CI, 0.977~0.998; p < 0.001; Fig. 3), and the sensitivity and specificity were 97% and 95%, respectively (Table 1). The cut-off value (0.607) was determined using 47 negative serum samples. Comparison of the two methods revealed a highly significant correlation (R² = 0.717) between the results for the ELISA developed in the study and commercial kit (Fig. 4).