Figure 3

Selective activation of pro-Canakinumab by MMP-9 treatment. Canakinumab or pro-Canakinumab (25 nM) were incubated with 4 μg recombinant MMP-9 for 0, 15, 30 and 60 min, respectively. (A) The light chain molecular weight of the Canakinumab or active and inactive pro-Canakinumab were detected with HRP-conjugated anti-human IgG Fab Ab by Western blot and (B) the IL-1β binding ability of Canakinumab and pro-Canakinumab with or without MMP-9 treatment were analyzed by IL-1β-coated ELISA (n = 3). (C) The IL-1β antigen coated 96-well plate was also incubated with different concentrations of Canakinumab (blue circles), pro-Canakinumab (green upward triangle), Canakinumab pre-incubated with MMP-9 (red squares) or pro-Canakinumab pre-incubated with MMP-9 (pink downward triangles), respectively, and then the IL-1β binding ability was analyzed by ELISA. The percentage of mean absorbance values (405 nm) of triplicate determinations are shown (n = 3). The bars indicate the SD.