Europium labeled heregulin beta

Example 5

Binding of HRG to HER3-ECD in the Presence of HER3 Antibody (ELISA)

A Streptavidin-coated 96-well plate was incubated at 4° C. with cell culture supernatant containing SBP-tagged HER3-ECD. On the next day the wells were washed three times with washng buffer (PBS+0.05% Tween-20) and blocked with PBS containing 1% BSA for one hour. After another three washes with washing buffer, 40 μl antibody solution (in Delfia Binding Buffer) was added to each well as a 2× stock of the desired final concentrations (10⁻³ to 10³ nM, alternatively 10⁻⁴ to 10² nM). Immediately 40 μl of 20 nM Europium-labeled Heregulin-beta (PeproTech, Cat. #100-03) was added to achieve a final concentration of 10 nM. The plates were incubated on a shaker at room temperature for two hours. Following three washes with Delfia Wash Buffer, Delfia Enhancement Solution was added and incubated on a shaker for 15 minutes (light protected). Finally, the plates were measured in a Tecan Infinite F200 reader using a time-resolved fluorescence measurement protocol. The binding of anti-HER3 antibodies M-08-11, M-43-01 and M-46-01 can promote/induce binding of HRG to HER3-ECD until a plateau is reached at a signal of 200% (for M-08-11 and M-43-01) and 330% (for M-46-01). M-33 serves as an Isotype control and shows concentration-independent values of approximately 100%. Results are shown in FIGS. 15A and B.

Example 5

Binding of HRG to HER3-ECD in the Presence of HER3 Antibody (ELISA)

A Streptavidin-coated 96-well plate was incubated at 4° C. with cell culture supernatant containing SBP-tagged HER3-ECD. On the next day the wells were washed three times with washing buffer (PBS+0.05% Tween-20) and blocked with PBS containing 1% BSA for one hour. After another three washes with washing buffer, 40 μl antibody solution (in Delfia Binding Buffer) was added to each well as a 2× stock of the desired final concentrations (10⁻³ to 10³ nM, alternatively 10⁻⁴ to 10² nM). Immediately 40 μl of 20 nM Europium-labeled Heregulin-beta (PeproTech, Cat. #100-03) was added to achieve a final concentration of 10 nM. The plates were incubated on a shaker at room temperature for two hours. Following three washes with Delfia Wash Buffer, Delfia Enhancement Solution was added and incubated on a shaker for 15 minutes (light protected). Finally, the plates were measured in a Tecan Infinite F200 reader using a time-resolved fluorescence measurement protocol. The binding of M-05-74 (named M-074 in FIG. 14) can promote binding of HRG to HER3-ECD until a plateau is reached at a signal of 650. Results are shown in FIG. 14.

Example 5

Binding of HRG to HER3-ECD in the Presence of HER3 Antibody (ELISA)

A Streptavidin-coated 96-well plate was incubated at 4° C. with cell culture supernatant containing SBP-tagged HER3-ECD. On the next day the wells were washed three times with washing buffer (PBS+0.05% Tween-20) and blocked with PBS containing 1% BSA for one hour. After another three washes with washing buffer, 40 μl antibody solution (in Delfia Binding Buffer) was added to each well as a 2× stock of the desired final concentrations (10⁻³ to 10³ nM, alternatively 10⁻⁴ to 10² nM). Immediately 40 μl of 20 nM Europium-labeled Heregulin-beta (PeproTech, Cat. #100-03) was added to achieve a final concentration of 10 nM. The plates were incubated on a shaker at room temperature for two hours. Following three washes with Delfia® Wash Buffer, Delfia® Enhancement Solution was added and incubated on a shaker for 15 minutes (light protected). Finally, the plates were measured in a Tecan Infinite F200 reader using a time-resolved fluorescence measurement protocol. The binding of M-05-74 (named M-074 in FIG. 14) can promote binding of HRG to HER3-ECD until a plateau is reached at a signal of 650. Results are shown in FIG. 14.