Table 1.
Sequences of the overlapping peptides derived from JEV NS1 (SA14-14-2 strain, GenBank AF315119).
Figure 1.
SDS-PAGE and Western blotting analysis of JEV NS1expression.
A, SDS-PAGE analysis of recombinant MBP-NS1 protein. MBP-NS1 is approximately 90 kDa. B, Western blotting analysis of recombinant NS1 protein. The MBP-NS1 band was visualized with TMB membrane substrate.
Table 2.
Characteristics of MAbs against JEV NS1.
Figure 2.
Primary identification of epitopes with MAbs.
Five epitopes of JEV NS1 protein were mapped with MAbs by ELISA. Cell culture medium was used as the MAb control. Mouse anti-sera against JEV NS1 and normal mouse sera were used as positive and negative controls, respectively. Each bar indicates antibody reactivity as determined by the mean absorbance.
Figure 3.
Fine-mapping of the epitopes by ELISA with MAbs.
Epitope-containing peptides were truncated from the carboxy and amino terminals. After the truncated peptides were expressed as a GST fusion protein, they were probed with MAbs by ELISA respectively. The deduced minimal unit of each linear B-cell epitope is underlined.
Table 3.
Sequence alignment of the defined epitopes corresponding to NS1 amino acid residues in JEV isolates.a
Figure 4.
Identification of virus-specific epitopes with MAbs.
Sequences were aligned between the identified linear epitopes and the homologous regions of WNV, MVEV and DENV (1–4). The virus strains listed in this figure were selected as representative strains. The homologous peptides from WNV and MVEV epitopes were expressed and analyzed by ELISA using the correspond MAbs. All MAbs and anti-sera against JEV NS1 did not cross-react with the homologous peptides of WNV. For MVEV, only peptides 9 and 43 showed weak reactivity with MAb 8B5 and 3H11.
Figure 5.
The JEV NS epitopes did not react with anti-WNV and anti-DENV sera.
The identified epitope fusion proteins were used as coating antigen in an indirect ELISA. The reactivity of the epitope fusion proteins with anti-WNV and anti-DENV sera was assessed. Normal mouse serum was used as a control.