Toward a deeper understanding of dengue: novel method for quantification and isolation of envelope protein epitope-specific antibodies

The dengue viruses (DENV) envelope (E) protein is the main target of the antibody (Ab) response. Abs target different epitopes on the E-protein, including sE-dimer, E domain III (EDIII), and fusion loop (FL). Anti-EDIII Abs are mainly serotype-specific, whereas anti-FL Abs can induce antibody-dependent enhancement (ADE) in vitro. Abs targeting sE-dimer epitopes can cross-neutralize different DENV serotypes. However, the involvement of each Ab subset in disease pathogenicity and/or protection remains unclear. We aimed to optimize the quantification and purification of DENV E-protein epitope-specific Abs from human samples. C-terminal biotinylated DENV2 E recombinant proteins (EDIII, soluble E [sE], and sE-dimer) were coupled to color-coded magnetic microspheres for a multiplex immunoassay (MIA), testing different antigen concentrations. Assay performance was evaluated using well-characterized anti-DENV monoclonal antibodies (mAbs) and total IgG from DENV seronegative and seropositive human plasma. Specific FL epitopes were blocked with mouse mAb clone 4G2 to quantify anti-FL- and sE-dimer-specific Abs, measuring antigen-antibody reactions as median fluorescence intensity (MFI). For isolation of E-protein epitope-specific antibodies, sE-proteins were conjugated to streptavidin resin beads. Total IgG from human plasma was incubated with immobilized EDIII to elute anti-EDIII Abs. The flow-through was incubated with sE-dimer resin beads to elute sE-dimer specific Ab enriched fraction, and the flow-through was applied to immobilized sE to elute anti-FL Abs. In conclusion, we have developed a serological assay to detect E-protein epitope-specific Abs in DENV-infected humans. Additionally, we successfully isolated anti-EDIII, anti-FL, and an enriched fraction of sE-dimer specific Abs from human samples.