High performance dengue virus antigen-based serotyping-NS1-ELISA (plus): A simple alternative approach to identify dengue virus serotypes in acute dengue specimens

Abstract

Dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV). Four different serotypes (DENV1-4) co-circulate in dengue endemic areas. The viral RNA genome-based reverse-transcription PCR (RT-PCR) is the most widely used method to identify DENV serotypes in patient specimens. However, the non-structural protein 1 (NS1) antigen as a biomarker for DENV serotyping is an emerging alternative method. We modified the serotyping-NS1-enzyme linked immunosorbent assay (stNS1-ELISA) from the originally established assay which had limited sensitivity overall and poor specificity for the DENV2 serotype. Here, four biotinylated serotype-specific antibodies were applied, including an entirely new design for detection of DENV2. Prediction of the infecting serotype of retrospective acute-phase plasma from dengue patients revealed 100% concordance with the standard RT-PCR method for all four serotypes and 78% overall sensitivity (156/200). The sensitivity of DENV1 NS1 detection was greatly improved (from 62% to 90%) by the addition of a DENV1/DENV3 sub-complex antibody pair. Inclusive of five antibody pairs, the stNS1-ELISA (plus) method showed an overall increased sensitivity to 85.5% (171/200). With the same clinical specimens, a commercial NS1 rapid diagnostic test (NS1-RDT) showed 72% sensitivity (147/200), significantly lower than the stNS1-ELISA (plus) performance. In conclusion, the stNS1-ELISA (plus) is an improved method for prediction of DENV serotype and for overall sensitivity. It could be an alternative assay not only for early dengue diagnosis, but also for serotype identification especially in remote resource-limited dengue endemic areas.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method

“…Roltgen et al (2018) developed an ELISA capable of distinguishing four DENV serotypes [ 48 ]. Similar ELISAs were also developed by other groups [ 40 , 41 ]. ELISAs have advantages in quantification, but immunochromatographic tests have the advantage of convenience for individual tests of a small number of samples [ 33 ].…”

“…The prevalence of these amino acid sequence differences in various geographical areas should also be determined. Previous studies showed variations in sensitivity and specificity depending on the collection date after the onset of illness, viral characteristics, and heterotypic DENV infection in NS1 DENV-specific detection using clinical specimens [ 40 , 41 , 42 , 43 , 44 ]. These factors might have also played a role in the cross-reactivity observed in the present study.…”

Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

“…The serotyping NS1 microplate ELISA was developed as described [ 22 ]. Briefly, each plasma sample (1:5 dilution) was added to four wells pre-coated with the pan-serotype capture antibody (2E11).…”

“…Biotinylated serotype-specific antibodies were added to each well separately. In some cases, an additional assay using the antibody pair 2E11/5F3 (for DENV1 and DENV3 subcomplex) was used to increase sensitivity of the assay [ 22 ]. Positive samples were classified when the assay OD reading exceeded twice the value of the negative plasma control.…”

“…Within the past decade, microplate DENV serotyping-NS1 ELISAs have also been developed as in-house assays, but prediction of some serotypes was still inaccurate [ 12 , 13 , 14 ]. Our recent modified serotyping-NS1 microplate ELISA (plus) showed perfect concordance in four serotypes identification in dengue patients’ specimens comparing to RT-PCR [ 22 ], indicating that immunoassay methods are capable of accurate serotyping. An ideal DENV diagnostic tool would need to be as sensitive and specific as possible, be able to be interpreted in real-time, should be easy to perform without investment in laboratory instruments, cheap to manufacture, and capable of classifying DENV serotypes.…”