Table 1.
Monoclonal antibody (mAb) designation and characterization.
Figure 1.
Western blot analysis of mAbs raised against the homologous DENV serotype.
Purified gamma-irradiated DENV-1 BR/01-MR (A), DENV-2 BR/01-01 (B), and DENV-3 290-02 (C) were subjected to 13% SDS-PAGE and electroblotted onto nitrocellulose membranes. Proteins were stained with the mAbs, followed by anti-mouse IgG conjugated to alkaline phosphatase. The flavivirus-specific mAb 4G2 and a non-correlated mAb that binds to hantavirus nucleoprotein (clone 572/7A) were used as positive (+) and negative (−) controls, respectively.
Figure 2.
Representation of the reactivities of major groups of monoclonal antibodies.
Indirect immunofluorescence of C6/36 cells uninfected (MOCK) or infected with DENV-1 (BR/90), DENV-2 (ICC 266), DENV-3 (290-02) and DENV-4 (TVP 360) isolates. Cells were fixed in methanol:acetone and stained with different mAbs, followed by Alexa-Fluor 488-conjugated anti-mouse immunoglobulin. Monoclonal antibody 4G2 and a non-correlated anti-hantavirus mAb (clone 572/7A) were used as positive and negative controls, respectively. Distinct groups of mAbs were raised against DENV: 1) group-specific (D3 424/8G); 2) subcomplex-specific (Anti-DENV-1, anti-DENV-3 and anti-DENV-4; clone D1 463/G6/H2); and 3) serotype-specific (anti-DENV-3 D3 290/4C/G9) mAbs. Images were produced in a Leica AF6000 Modular System. Scale bars are 30 µm.
Figure 3.
Cross-reactivity of mAbs D3 424/8G and D3 863/G7/H7 against WNV, SLEV and YFV.
Vero E6 cells were infected with WNV (A), whereas Huh7.5 cells were infected with YFV and SLEV (B). Cells were fixed in methanol:acetone and stained with mAbs, followed by Alexa-Fluor 488-conjugated anti-mouse immunoglobulin. Monoclonal antibody 4G2 and a non-correlated anti-hantavirus mAb (572/7A) were used as positive and negative controls, respectively. Images were obtained with a Leica AF6000 Modular System. Scale bars are 30 µm.
Table 2.
Cross-reactivity of anti-dengue virus monoclonal antibodies against YFV, SLEV, WNV and VEEV.
Figure 4.
Application of antibodies to the development of MAC-ELISA.
HRP-conjugated D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H mAbs were used in an in-house MAC-ELISA assay to detect anti-dengue virus IgM in the sera of infected (N = 22) and non-infected patients (N = 24).