A novel anti-NS2BNS3pro antibody-based indirect ELISA test for the diagnosis of dengue virus infections

Abstract

Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.