Fig 1.
Performance of ELISA Maxisorp® and Polysorp® microplates in the in house ELISA assay.
Plates were sensitized with recombinant capsid protein (5 μg/well) and reacted with 5 positive and 5 negative samples previously tested with the commercial kit. Significant differences (p<0.05) on the OD obtained with the same set of serum are indicated by asterisk (*).
Table 1.
Performance of ELISA microplates.
Plates were sensitized with recombinant ORF-2 protein and tested with known positive (P) and negative (N) serum to determine the P/N relation and the percentile of the coefficient of variation (CV%). The index value was obtained by dividing the result of P/N by the CV%.
Fig 2.
Optimal antigen concentration for the in house ELISA.
Recombinant capsid protein (5 μg) was twofold serially diluted on Maxisorp® microplate. Serum samples, previously determined to be positive (n = 5) or negative (n = 5) were evaluated. The data is represented as the mean ± SEM of the OD of each antigen dilution. The arrow indicates the optimal antigen concentration.
Fig 3.
Determination of the primary serum dilution.
Human serum samples known to be positive (n = 5) or negative (n = 5) were diluted as indicated in the figure and tested for their ability to bind the recombinant capsid protein (5 μg/well) adsorbed onto Maxisorp® ELISA microplates. The results are expressed as the mean ± SEM of the OD obtained at each serum dilution. The asterisk (*) indicates the lowest serum dilution with significant difference (p<0.05) to the immediately higher dilution within positive or negative samples.
Fig 4.
Receiver Operating Characteristic (ROC) analysis.
The ROC curve was generated using the results obtained by analyzing 50 negative human serum samples and 63 positive human serum samples by the iELISA. The area under the ROC curve was of 0.9955.
Table 2.
Agreement between the performance of the in house ELISA and the RecomWell (Mikrogen®) in detecting anti-HEV IgG.
Eight seven serum samples were randomly selected amongst the samples used in this study and tested in duplicates with the commercial ELISA kit to determine the presence of anti-HEV antibodies.
Fig 5.
Prevalence of anti-HEV antibodies amongst blood donors.
The in house ELISA was performed using the ideal concentration of antigen and ideal serum dilution, with 780 blood samples. The results are expressed as the P/N ratio, as described in material and methods. Samples with a P/N ratio was ≥ 2.5. The cut-off value is represented by the dashed line.