Purified rNadA binds Chang cells in a time- and temperature dependent manner.

A and B. Chang cells were incubated with 200 µg/ml rNadA and then washed with PBS – 1% FBS. Binding was detected using anti-NadA 9F11 mouse mAb and Allophycocyanin-conjugated goat anti-mouse secondary antibody. Analysis was performed with Canto II instrument, and mean fluorescence intensity is reported (MFI). A: Binding of rNadA to cells for 30 minutes at the indicated temperatures, and using varying concentrations of the primary antibody B: Binding of rNadA was performed at 37°C for a period of time ranging from 1 minute to 2 hours as indicated. C: SE-HPLC profile of rNadA at room temperature. D: SE-HPLC profile of rNadA after heating period of 4 hrs at 37°C. E: SDS-Page of collected fractions. Black arrows in D indicate the fractions pooled.