Precise characterisation of monoclonal antibodies to the C-terminal regionof p53 protein using the PEPSCAN ELISA technique and a new non-radioactivegel shift assay

The development of human cancers is frequently associated with inactivation of the p53 tumour suppressor protein triggering cell cycle arrest or apopt osis in response to cellular stress. The p53 protein has been identified as a transcription factor with sequence-specific DNA binding properties. The DNA-binding activity is cryptic but can be modulated through the C-terminal region of the p53 protein by several different stimuli, including phosphor ylation by casein kinase II (CKII), protein kinase C (PKC) or binding of th e C-terminal monoclonal antibody PAb421. Monoclonal antibodies to the C-ter minal region of p53 protein are able to activate the latent form of p53 and induce binding to DNA. To characterise such antibodies, we used a combinat ion of the PEPSCAN ELISA procedure and a newly developed non-radioactive ge l shift assay. Monoclonal antibodies from the Bp53 series displayed higher affinities for the human, rat and mouse p53 proteins than did the conventio nal antibody PAb421. In addition, these antibodies were able to activate th e sequence-specific DNA binding functions in latent forms of p53 protein an d, in contrast to PAb421, they were able to recognise both PKC phosphorylat ed and PKC non-phosphorylated forms of p53 protein. Our monoclonal antibodi es recognising post-translationally modified target epitopes in the C-termi nal region of p53 protein might assist the development of more effective mo lecules for p53-based cancer therapy. (C) 2000 Elsevier Science B.V. All ri ghts reserved.