DENV type 4 Envelope protein (32 kDa)

The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC(50)) of these MAbs. Using PRNT as the reference and treating IC(50) values higher than 50 mu g/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R(2) = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV.

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.