Notch signalling occurs via direct cell-cell interactions and plays an important 
role in linking the fates of neighbouring cells. There are four different 
mammalian Notch receptors that can be activated by five cell surface ligands. 
The ability to inhibit specific Notch receptors would help identify the roles of 
individual family members and potentially provide a means to study and control 
cell differentiation. Anti-Notch antibodies in the form of single chain Fvs were 
generated from an antibody phage display library by selection on either the 
ligand binding domain or the negative regulatory region (NRR) of Notch1 and 
Notch2. Six antibodies targeting the NRR of Notch1 and four antibodies 
recognising the NRR of Notch2 were found to prevent receptor activation in 
cell-based luciferase reporter assays. These antibodies were potent, highly 
specific inhibitors of individual Notch receptors and interfered with endogenous 
signalling in stem cell systems of both human and mouse origin. 
Antibody-mediated inhibition of Notch efficiently down-regulated transcription 
of the immediate Notch target gene hairy and enhancer of split 5 (Hes5) in both 
mouse and human neural stem cells and revealed a redundant regulation of Hes5 in 
these cells as complete down-regulation was seen only after simultaneous 
blocking of Notch1 and Notch2. In addition, these antibodies promoted 
differentiation of neural stem cells towards a neuronal fate. In contrast to the 
widely used small molecule γ-secretase inhibitors, which block all 4 Notch 
receptors (and a multitude of other signalling pathways), antibodies allow 
blockade of individual Notch family members in a highly specific way. Specific 
inhibition will allow examination of the effect of individual Notch receptors in 
complex differentiation schemes regulated by the co-ordinated action of multiple 
signalling pathways.