Human her2 fc chimera

Example 64

The binding affinity of trastuzumab-coil-VM24 fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 μg/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH 7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH 7.4), various concentrations of trastuzumab IgG and trastuzumab-coil-VM24 fusion proteins are added to the plate for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added to the plate and the plate is incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluoregenic peroxidase substrate to each well.

Example 63

The binding affinity of trastuzumab-coil-hGCSF fusion proteins to Her2 receptor is examined by ELISA. Human Her2-Fc chimera (5 μg/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH 7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH 7.4), various concentrations of trastuzumab IgG and trastuzumab-coil-hGCSF fusion proteins are added to the plate for 2 hours of incubation at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added to the plate and the plate is incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluoregenic peroxidase substrate to each well.

Example 20

The binding affinity of trastuzumab-coil-exendin-4 fusion proteins to Her2 receptor is examined by ELISA Human Her2-Fc chimera (5 ug/mL) (R&D Systems) is coated on 96-well ELISA plate overnight at 4° C., followed by blocking with 1% BSA in PBS (pH7.4) for 2 hours at 37° C. After washing with 0.05% Tween-20 in PBS (pH7.4), varied concentrations of trastuzumab IgG (SEQ ID NOs: 19 and 22) and trastuzumab-coil-exendin-4 (SEQ ID NOs: 71 and 19) fusion proteins are added to incubate for 2 hours at 37° C. Subsequently, goat polyclonal anti-human kappa light chain antibody with HRP conjugate (Sigma) is added and incubated for 2 hours at 37° C. Wells are subsequently washed and binding affinities are examined on the basis of fluorescence intensity at 425 nm by adding fluoregenic peroxidase substrate to each well.