Table 1.

Characterization of Notch mAbs

Anti-Notch1 mAba% binding inhibition Notch1 (n = 3)b% binding inhibition Notch2 (n = 3)b% signaling inhibition Notch1 (n = 3)c% signaling inhibition Notch2 (n = 3)c% proliferation inhibition (n = 3)d
CloneEpitope (EGF repeats)hJAG1hDLL4hJAG1hDLL4hJAG1hDLL4hJAG1hDLL4hNotch1hNotch2
  • aEpitope specificity of Notch mAbs: mAbs were incubated with either EGF repeats 11 to 15 or 1 to 12 adsorbed on a plastic surface and binding was determined. The mAbs that recognized both receptor fragments were considered as EGF repeats 11 to 12 specific, whereas others were identified as EGF repeats 13 to 15–specific mAbs.

  • bThe HEK293 hN1 or hN2 cells were incubated with the control IgG or mAbs (10 μg/mL) for 1 hour followed by washing with DPBS and incubation with the saturating concentration of ligands (20 μg/mL) for 1 hour on ice. The cells were then incubated with antihuman Fc conjugated with fluorescein isothiocyanate antibody and binding was determined by flow cytometry. Ratio of median fluorescence intensity in the presence of control IgG and Notch1 IgG was calculated for percent inhibition.

  • cHEK293 hN1 or hN2 cells transfected with 12xCSL-Luc reporter plasmid were cultured for 36 hours on plates precoated with 20 μg/mL soluble Jagged1/Delta-like4 Fc in the presence of 10 μg/mL control IgG or anti-Notch1 IgG, and the luciferase activities were determined by dual luciferase assay. The ratio of firefly luciferase to Renilla luciferase was calculated for normalization. Ratio of normalized values for the control IgG to anti-Notch1 IgG was calculated for determining percentage of inhibition.

  • dHEK293 hN1, hN2, or vector-alone cells were cultured on Jagged1 Fc precoated plates in the presence of 10 μg/mL control IgG or anti-Notch1 mAb. BrdUrd incorporation was investigated by anti-BrdUrd–specific antibody in ELISA and percentage of inhibition was determined compared with control IgG.