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Abstract: Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin–streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20mg protein of ∼95% purity, from 1L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be ∼85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin–enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera (n =164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity. [ABSTRACT FROM AUTHOR]