RAPID AND SENSITIVE STREPTAVIDIN-BIOTIN AMPLIFIED FLUOROGENIC ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR DIRECT-DETECTION AND IDENTIFICATION OF DENGUE VIRAL-ANTIGENS IN SERUM

Each of the four serotypes of dengue viruses is responsible for a spec trum of illnesses that range from nonspecific febrile syndrome with go od prognosis to dengue haemorrhagic fever or dengue shock syndrome. De finite diagnosis of dengue is provided by the detection of virus in ac ute-phase sera of patients. Virus isolation can be accomplished with m osquito cell lines or mosquito inoculations. However, these methods ar e time consuming and labour intensive. The reverse-transcriptase polym erase chain reaction (RT-PCR) provides a potential means of rapid diag nosis but requires specialised facilities and equipment and is expensi ve. Therefore a rapid, simple, sensitive, and economical method for di rect detection of viral antigens in viraemic sera is needed for clinic al and epidemiological investigations. An amplified fluorogenic enzyme -linked immunosorbent assay (F-ELISA) is described for the detection a nd identification of dengue-3 viruses in serum specimens. This assay u tilizes biotinylated mouse IgG antibody directed against dengue antige ns captured by anti-dengue monoclonal antibody coated onto polystyrene microplate wells. It takes advantage of the high affinity of biotin f or the multivalent binding sites of streptavidin-labelled beta-galacto sidase, and combines the amplification effect of biotin-streptavidin i nteraction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing nonspecific bindi ng of proteins and enhancing the specific binding of antigens, F-ELISA was tested on 259 sera submitted routinely to our laboratory for conf irmation 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. (C) 1995 Wiley-Liss, Inc.