Comparison of the sensitivities of the WNV-NS1 ELISA and real-time RT-PCR for the detection of WNV-infected mice serum samples.

<p>Comparison of the sensitivities of the WNV-NS1 ELISA and real-time RT-PCR for the detection of WNV-infected mice serum samples.</p

The sensitivity of the double antibody sandwich ELISA using different capture and detection MAbs pairs.

<p>The detection of NS1 in culture supernatants of WNV-infected cells by different MAb pairs. The culture supernatants of WNV-infected cells were serially diluted 2-fold. Data points represent the mean ± standard deviation from three replicates. The most effective pairs were determined by the highest dilution of the OD₄₅₀ values.</p

Characteristics of MAbs against the NS1 protein of WNV.

a<p>Purified recombinant WNV-NS1 protein and DENV-NS1 were used as the coating antigen and were reacted with purified MAbs against the NS1 protein. The absorbance was measured at 450 nm: +, OD₄₅₀ = 0.5 to 1; ++, OD₄₅₀ = 1 to 2; +++, OD₄₅₀ = 2 to 3; OD₄₅₀>3.</p>b<p>The reactivity of each MAb was determined by IFA with WNV, DENVs, YFV, and JEV, and their IFA reactivity with normal cells were negative (data not shown). −, no reactivity; +, weak reactivity; ++, intermediate reactivity; +++, strong reactivity.</p>c<p>Not detected.</p><p>Characteristics of MAbs against the NS1 protein of WNV.</p

Evaluation of the sensitivity and specificity of the rWNV-NS1 antigen-capture ELISA.

<p>A, The NS1 standard curve as determined by the purified rWNV-NS1 antigen-capture ELISA. Various concentrations of WNV-NS1 and DENV1-4-NS1 proteins were analysed. BSA was used to establish the baseline. B, The detection of NS1 in WNV-infected cell culture supernatants and other closely related members of the flavivirus family, DENV, JEV, YFV, and TBEV, by the NS1 antigen-capture ELISA. Two-fold serially diluted supernatants from WNV-, four DENV serotype-, JEV-, YFV-, or TBEV-infected cells were subjected to the WNV-NS1 antigen-capture ELISA. Data points represent the mean ± standard deviation from three replicates. The cut-off value was set at twice the average value of the negative controls from three replicates. Positive OD450 values were only observed for the WNV-NS1 protein and WNV-infected cell supernatants.</p

The detection of NS1 in WNV-infected mice sera by the NS1 antigen-capture ELISA.

<p>The data represent the OD₄₅₀ of the serum samples at 1∶100 dilution. The dashed line represents the cut-off value, which is twice the mean value of the negative control. Each data point represents the mean OD₄₅₀ of duplicate assays. The results were considered positive if a sample yielded an OD₄₅₀ above the cut-off value. The cut-off value was set as twice the mean value of the negative control. Each data point represents the mean OD₄₅₀ of duplicate assays.</p

Schematic representation of tandem EDIII constructs.

<p>The amino acid sequence covering EDIII (298aa∼400aa) from each serotype was arranged sequentially, and (Gly₄Ser)₃ sequence was used to connect EDIII regions. Thioredoxin (Trx) was fused at the N-terminal to increase solubility and the C-terminal His-Patch was included for purificaiton through Ni-NTA agarose.</p

Neutralizing antibody responses in mice immunized with MixBiEDIII.

<p>Neutralization titers of MixBiEDIII immune sera at two weeks after the last immunization were measured by PRNT₅₀. The neutralizing antibodies against four DENV serotypes were significantly induced by MixBiEDIII compared with control group. Dotted line represents limits of detection.</p

Characterization of the tandem bivalent recombinant EDIII antigens.

<p>ELISA of purified EDIII proteins was performed using corresponding monoclonal antibody against different serotype of DENV. Trx protein was control. The cut-off for the ELISA is shown by a dotted line.</p

Protection of anti-MixBiEDIII sera against all four serotypes DENV.

<p>The suckling mice were injected intracranially (IC) with the sera-virus mixture, and mortality was monitored for 21 days. Data represent the mean±SD.</p>a<p>P<0.05 for MixBiEDIII sera group vs. control sera group, by Log-Rank analysis.</p

IgG antibody responses in mice immunized with MixBiEDIII.

<p>The total lgG titers of immune sera from mice immunized with MixBiEDIII at two weeks after the last immunization were measured by ELISA. The lgG titers of immune sera against four DENV serotypes from mice immunized with MixBiEDIII were significant higher than control group. Dotted line represents limits of detection.</p