Complement-Mediated Neutralization of Dengue Virus Requires Mannose-Binding Lectin

Avirutnan, Panisadee; Hauhart, Richard E.; Marovich, Mary A.; Garred, Peter; Atkinson, John P.; Diamond, Michael S.

MBL-DENV capture ELISA. MBL binding to DENV was evaluated using a virus capture ELISA with MAb-coated wells on microtiter plates as described previously (25) with the following modifications: the wells on microtiter plates were adsorbed with an anti-DENV prM protein-specific MAb 2H2 (55) or an isotype control (IgG2a) anti-hepatitis C virus E2 MAb H77.16 (56) (10 g/ml in PBS) at 4°C overnight. DENV-2 stocks were diluted in DMEM to 5 ϫ 10 5 PFU/ml and added to wells. The wells on the plates were washed and then incubated with increasing concentrations of purified human recombinant MBL diluted in binding buffer (20 mM Tris-HCl [pH 7.4], 0.05% Tween 20, 0.1% [wt/vol] BSA, 1 M NaCl, and 10 mM CaCl 2 ). The wells on the plates were washed, incubated sequentially with biotin-conjugated mouse anti-human MBL MAb (Cedarlane), horseradish peroxidase (HRP)-conjugated streptavidin, and developed with 3,3=,5,5=-tetramethylbenzidine (TMB) substrate (DAKO). The optical density (OD) at 450 nm was determined by a 96-well plate reader (Genio Pro; Tecan Instruments). In some experiments, captured DENV-2 was incubated with human serum (1:4 dilution) instead of purified recombinant human MBL. For a negative control, purified human MBL was diluted in 20 mM Tris-HCl (pH 7.4) and 150 mM NaCl supplemented with 40 mM EDTA or supplemented with 100 g/ml mannan. In parallel experiments, captured DENV virions were detected by biotinylated anti-DENV E-protein-specific MAb 4G2 (55) (1 g/ml), followed by HRP-conjugated streptavidin (2 g/ml).