The binding assay was performed as described previously [12] . In brief, 96-well flat-bottom polystyrene plates (Corning-Costar) were precoated with 100 lL/well of 10 2 , 10 3 ,10 4 , and 10 5 TCID 50 UV-irradiated influenza viruses or phosphate-buffered saline (PBS). After incubation at room temperature overnight, wells were blocked for 2 hours at room temperature with 1% bovine serum albumin (BSA) in PBS with 0.05% sodium azide. Different concentrations of recombinant human MBL (rhMBL) (0, 0.5, 2, 6, or 8 lg/mL), which was kindly provided by Dr K. Takahashi (Laboratory of Developmental Immunology, Harvard Department of Pediatrics, Massachusetts General Hospital, Boston), were added and incubated overnight at 4°C. Then 100 lL of 0.2 lg/mL biotinylated monoclonal anti-MBL antibody (HYB131-01, Antibody Shop) diluted in PBS with 1% BSA was added into each well. Bound antibody was detected by using horseradish peroxidase-conjugated streptavidin and tetramethybenzidine substrate solution (R&D Systems). The binding of MBL to influenza virus was evaluated by the absolute absorbance values measured at 450 nm (A 450 ).