Recombinant Llama Anti-DLL4 Single Domain Antibody (HPAB-1513-FY)

The blocking capacity of the VHHs is evaluated in a human DII4 - human Notch1/Fc blocking ELISA. In brief, 1 μg/mL of human Notch1/Fc chimera is coated in a 96-well MaxiSorp plate. A fixed concentration of 15 nM biotinylated human DII4 is preincubated with a dilution series of the VHH for 1 hour, after which the mixture is incubated on the coated Notchi receptor for an additional hour. Residual binding of biotinylated human DII4 is detected using horseradish peroxidase (HRP) conjugated extravidin.

The blocking capacity of the VHHs is evaluated in a human DII4 - human Notch1/Fc blocking ELISA. In brief, 1 μg/mL of human Notch1/Fc chimera is coated in a 96-well MaxiSorp plate. A fixed concentration of 15 nM biotinylated human DII4 is preincubated with a dilution series of the VHH for 1 hour, after which the mixture is incubated on the coated Notchi receptor for an additional hour. Residual binding of biotinylated human DII4 is detected using horseradish peroxidase (HRP) conjugated extravidin.

Recombinant mouse DII4 is coated overnight at 4°C at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1 % in PBS). VHHs are applied as dilution series and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, MO, USA).

Recombinant mouse DII4 is coated overnight at 4°C at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1 % in PBS). VHHs are applied as dilution series and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, MO, USA).

Cynomolgus DII4 expressing HEK293 cells (transient or stable transfection) are used for a titration binding experiment of the VHHs. After a 30 minutes incubation on ice, all samples are washed and detection is performed by applying anti-c-myc~Alexa647 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Human and mouse DII4 overexpressing HEK293 cells are taken as reference. The mean MCF value is determined on the FACS Array and used for calculation of the EC50 value.

Cynomolgus DII4 expressing HEK293 cells (transient or stable transfection) are used for a titration binding experiment of the VHHs. After a 30 minutes incubation on ice, all samples are washed and detection is performed by applying anti-c-myc~Alexa647 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Human and mouse DII4 overexpressing HEK293 cells are taken as reference. The mean MCF value is determined on the FACS Array and used for calculation of the EC50 value.