Rapid and sensitive streptavidin‐biotin amplified fluorogenic enzyme‐linked immunosorbent‐assay for direct detection and identification of dengue viral antigens in serum

Abstract

Each of the four serotypes of dengue viruses is responsible for a spectrum of illnesses that range from nonspecific febrile syndrome with good prognosis to dengue haemorrhagic fever or dengue shock syndrome. Definite diagnosis of dengue is provided by the detection of virus in acute-phase sera of patients. Virus isolation can be accomplished with mosquito cell lines or mosquito inoculations. However, these methods are time consuming and labour intensive. The reverse-transcriptase polymerase chain reaction (RT-PCR) provides a potential means of rapid diagnosis but requires specialised facilities and equipment and is expensive. Therefore a rapid, simple, sensitive, and economical method for direct detection of viral antigens in viraemic sera is needed for clinical and epidemiological investigations. An amplified fluorogenic enzyme-linked immunosorbent assay (F-ELISA) is described for the detection and identification of dengue-3 viruses in serum specimens. This assay utilizes biotinylated mouse IgG antibody directed against dengue antigens captured by anti-dengue monoclonal antibody coated onto polystyrene microplate wells. It takes advantage of the high affinity of biotin for the multivalent binding sites of streptavidin-labelled beta-galactosidase, and combines the amplification effect of biotin-streptavidin interaction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing non-specific binding of proteins and enhancing the specific binding of antigens, F-ELISA was tested on 259 sera submitted routinely to our laboratory for confirmation of dengue diagnosis. The sensitivity of the F-ELISA was 90%, the specificity was 99% and the agreement rate was 98% between F-ELISA and virus isolation results.