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

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.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion.

BACKGROUND: Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. OBJECTIVE: To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. METHODS: VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. RESULTS: Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. CONCLUSIONS: A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.