Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay

Abstract

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-α) in serum samples of children infected with dengue virus ( n=99) and healthy controls ( n=41). The minimum detectable concentration of TNF-α by the BS-ELISA was 3.3 pg/ml. The mean TNF-α level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44±42.0 pg/ml). Lower levels were found in DHF grade I (28.44±42.7 pg/ml), DHF grade II (24.21±25.4 pg/ml) and dengue fever (DF) (14.10±24.0 pg/ml). TNF-α in the sera of DF and DHF patients could be detected on days 2–6 after the onset of fever, the high level occuring on day 5. TNF-α was detected in 41.4% (24.01±35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2±15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-α than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-α than DF patients ( P-value=0.020) but no difference in the TNF-α levels from sera of DHF grades I–III patients was observed ( P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-α in serum samples of DF and DHF patients. The TNF-α levels might be associated with dengue virus infection and related to disease severity of DHF.