Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of ß-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.

Performance characteristics of the VIDAS® ANTI-HEV IgM and IgG assays.

BACKGROUND: Several unautomated anti-HEV diagnostic tests are presently available. OBJECTIVE: We have evaluated the performance of the new automated VIDAS® ANTI-HEV IgM and IgG assays. STUDY DESIGN: We assessed the reproducibility and cross-reactivity of both VIDAS assays and the analytical sensitivity and linearity of the VIDAS IgG assay. We also tested the VIDAS and comparator assays Wantai IgG and IgM on immunocompetent and immunocompromised patients. Data were analysed according to the infectious profile, with samples from viremic phase (HEV RNA/IgM positive) and post-viremic phase (HEV RNA negative, IgM positive) infections, and uninfected patients (HEV RNA/IgM negative). RESULTS: Within-run reproducibility was <10% and between-run reproducibility was <12% for both assays. We found no cross-reactivity, except for the VIDAS IgG assay in some patients with HBV (1/10) or malaria (3/23) infections and for the VIDAS IgM assay in some HIV-infected patients (1/10). The VIDAS IgG assay was linear over 0.10-10.0 U/mL. Analytical sensitivity of the IgG assay was 0.71 IU/ml (probit analysis). The clinical sensitivity of the VIDAS IgM assay was 97.65% for viremic samples (83/85) and 59.15% (42/71) for post-viremic samples from immunocompetent patients. It was 78.95% (45/57) for acute phase samples and 77.78% (28/36) for post-viremic samples from immunocompromised patients. Specificity was excellent (>99%) in both populations. CONCLUSION: The analytical and clinical performance of the new VIDAS® ANTI-HEV assays was excellent. These rapid, automated assays for detecting HEV antibodies will strengthen the arsenal for diagnosing HEV infections.