65r 510phrp

Example 9

This example shows the blocking of the interaction between recombinant HGF and recombinant c-Met by anti-c-Met antibody A1 and its optimized versions. Inhibition of ligand binding to its receptor prevents activation. In this example, an ELISA was used to determine the concentration at which 50% of the ligand/receptor binding was blocked by the antibodies (IC50). Here, recombinant c-Met extracellular domain (R&D Sytems cat #358-MT-100/CF) was immobilized to the ELISA plate followed by blocking with SuperBlock (Scytek, Cat #AAA500). The antibodies were then added to the plate in an 8-point, 4-fold serial dilution. After incubation for 1 hr and washes, HGF (R&D Systems cat #294-HG-005/CF) was added to the plates at a final concentration of 0.15 nM. HGF binding to c-Met is detected using a biotinylated anti-HGF antibody (R&D cat #BAF294) followed by Streptavidin-HRP (Fitzgerald cat #65R-510PHRP). OD450 was plotted against antibody concentration and non-linear regression (GraphPad Prism) is used to determine the IC50 for each antibody (FIG. 10). The data is shown as mean OD450+/−SEM and the IC50 values shown are in nM.

Example 8

This example shows the blocking of the interaction between recombinant HGF and recombinant c-Met by anti-c-Met antibody E1 and its optimized versions. Inhibition of ligand binding to its receptor prevents activation. In this example, an ELISA was used to determine the concentration at which 50% of the ligand/receptor binding was blocked by the antibodies (IC50). Here, recombinant c-Met extracellular domain (R&D Sytems cat #358-MT-100/CF) was immobilized to the ELISA plate followed by blocking with SuperBlock (Scytek, Cat #AAA500). The antibodies were then added to the plate in an 8-point, 4-fold serial dilution. After incubation for 1 hr and washes, HGF (R&D Systems cat #294-HG-005/CF) was added to the plates at a final concentration of 0.15 nM. HGF binding to c-Met was detected using a biotinylated anti-HGF antibody (R&D cat #BAF294) followed by Streptavidin-HRP (Fitzgerald cat #65R-510PHRP). OD450 was plotted against antibody concentration and non-linear regression (GraphPad Prism) was used to determine the IC50 for each antibody (FIG. 9). The data is shown as mean OD450+/−SEM and the IC50 values shown are in nM.