Development and validation of dengue virus envelope protein domain III IgG antibody enzyme-linked immunosorbent assay

Abstract

Introduction

The global incidence of Dengue virus (DENV) infections has significantly increased in recent decades, becoming a public health emergency of global concern. Diagnostic tools for DENV infection include detection of the virus, viral RNA, or viral antigens, which are usually cumbersome or require sophisticated medical facilities and trained personnel. Serological tests are the most widely used methods to detect dengue infection due to low cost and operational simplicity. The detection of antibodies (IgM and IgG) in the blood of an infected individual is an indirect method of diagnosing DENV and IgG can be used as long-term detection. The aim of this work was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-DENV IgG antibodies, and to determine its usefulness in the diagnosis and seroepidemiological survey of DENV.

Materials and methods

The production of the antigen was carried out using the tetravalent DENV E protein-based provided by Trebe Biotech. The recombinant baculovirus was obtained using the Bac to Bac® baculovirus expression system. The recombinant tetravalent dengue antigen was expressed in insect larvae, purified by IMAC and identified by SDS-PAGE and western blot analysis. The system used in this work has the advantage of using insect pests as biofactories. Recombinant protein production is high-yield and production times are short. Furthermore, it has no product size limit and is highly flexible to sequence changes or the emergence of new serotypes. An indirect ELISA was developed using purified tetravalent DENV antigen immobilized on polystyrene microplates; human sera samples from individuals with no history of DENV infection (n = 22) and human sera samples from individuals with clinical history and diagnosed of DENV infection (n = 23) were then incubated. Immune complexes were revealed with anti-human IgG-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. In order to establish DENV-IgG seroprevalence, the developed ELISA was applied to analyze samples from blood donor centers from different geographic regions of the country (n = 504) and normal human sera (n = 17).

Results

The efficiently produced recombinant dengue antigen was used for the development and validation of a high sensitivity (91.30 %) and specificity (90.91 %) indirect ELISA for the detection of anti-DENV IgG antibodies. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from normal control individuals and patients with DENV (AUC = 0.9901); the cut-off value was established at 2 SDs. In the assessment of seroprevalence levels of anti-DENV antibodies, 33 out of 504 analyzed samples (6.55 %) tested positive using our developed ELISA.

Conclusions

This technique is rapid, cost-effective, and easy to use, making it suitable for low or medium complexity laboratories, especially when compared to molecular or culture-based testing. This confirms that the assay is both robust and reliable for diagnosing DENV infection, making it suitable for resource-limited areas.