Fluorescent antibody techniques applied to the identification of dengue virus in infected tissue

We evaluated an indirect fluorescent antibody (FA) method for the detection of dengue virus antigen in infected mouse tissues. The biotin-avidin system [unlabeled antiviral antibody, biotinyl-anti-IgG and fluorescein conjugated avidin D(biotin-avidin system)] theoretically enhances the sensitivity of the FA method by amplifying the number of fluorescein particles attached indirectly to antigen. Using antibody endpoint titers in dengue-infected suckling mouse brain as an assay for sensitivity, we compared this three-step technique with the standard direct and two-step indirect FA techniques. Comparative tests were done on frozen sections of mouse brains with infectivity titers between 4.5 and 8.3 log10 LLC-MK2 cell PFU/g. Antibody endpoint titers with the biotin-avidin system were 2- to 8-fold higher than those obtained with the indirect and direct fluorescent antibody systems. The biotin-avidin system may be useful for rapid postmortem diagnosis of some fatal dengue hemorrhagic fever-dengue shock syndrome cases and perhaps also for early diagnosis of dengue by examination of leukocytes or biopsy material.