Monoclonal antibody to dengue capsid protein: its application in dengue studies

Abstract

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.

Figure 1

Immunofluorescence staining with 8H8 mAb of dengue infected cells with Den-2 A15 strain. C6/36-HT cells were infected with Den-2 A15. At 6 (a), 8 (b), 12 (c), 48 (d) and 72 (e) h p.i, the infected cells were reacted 8H8 mAb followed with an FITC-conjugated goat anti-mouse antibody and then examined under a fluorescence microscope. Cytoplasmatic and nuclear staining of infected cells are indicated as C and N, respectively. C6/36-HT cells infected with Den-2A15 reacted with anti-Den-2 polyclonal antibody at 72 h p.i was used as a positive control (f) and non-infected C6/36-HT cells were used as negative control (g).

Figure 2

Immunofluorescence staining with 8H8 mab of dengue infected cells. C6/36-HT cells were infected with samples 57 (a–c) and 59 (d–f) from dengue acute patients. At 6 (a), 12 (b), 72 (c) h pi, for sample 57 and 12 (d), 24 (e), 72 (f) h pi for sample 59, the infected cells were reacted 8H8 mab followed with an FITC-conjugated goat anti-mouse antibody and then examined under a fluorescence microscope. Cytoplasmatic and nuclear staining of infected cells is indicated as C and N, respectively. Samples from healthy individuals as negative control (g).

Figure 3

Staining of Dengue capsid protein in suckling mice brain at 24 (a), 48 (b) and 72 h (c) pi Uninfected suckling mouse brain as negative control (d).

Figure 4

Reactivity of 8H8 mab in the antigen-capture enzyme-linked immunosorbent assay (ELISA) format. The ability to capture the Dengue-2 recombinant protein (Den-2-rCP) of the monoclonal antibody was evaluated at various concentrations of Den-2-rCP.