Critical residues of epitopes recognized by several anti-p53 monoclonal antibodies correspond to key residues of p53 involved in interactions with the mdm2 protein

The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These antibodies could, therefore, represent powerful tools for the study of p53 regulation. (C) 2000 Elsevier Science B.V. All rights reserved.