UNCORRECTED PROOF JIM8660 10 Journal of Immunological Methods 1 (2000) 000–000 4 www.elsevier.nl / locate / jim 12 Critical residues of epitopes recognized by several anti-p53 13 monoclonal antibodies correspond to key residues of p53 14 involved in interactions with the mdm2 protein a, a b * 15 Jean-Michel Portefaix , Sabine Thebault , Florence Bourgain-Guglielmetti , a a a a 16 Maguy Del Rio , Claude Granier , Jean-Claude Mani , Isabelle Navarro-Teulon , a b a 17 Michel Nicolas , Thierry Soussi , Bernard Pau a ˆ 18 CNRS UMR9921, CRLC Val d’ Aurelle / Bat Recherche, Rue de la Croix Verte, 34298 Montpellier Cedex 5, France b 19 CNRS UMR218, Institut Curie, Pavillon Trouillet-Rossignol, 26 Rue d’ Ulm, 75231 Paris Cedex 5, France 20 Received 7 April 1999; received in revised form 16 September 1999; accepted 18 October 1999 21 22 Abstract 23 The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we 24 analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, 25 mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the 26 N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these 27 mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical 28 residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters 29 using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic 30 characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction 31 of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These 32 antibodies could, therefore, represent powerful tools for the study of p53 regulation.  2000 Elsevier Science B.V. All 33 rights reserved. 34 Keywords: p53; mdm2; Monoclonal antibody; Spot; Surface plasmon resonance 35 36 1. Introduction ptotic or growth arrest pathways in proliferating cells 39 (Hartwell and Kastan, 1994). Among the various 40 37 The p53 protein plays a crucial role in the cellular biochemical activities exerted by the p53 protein, 41 38 response to DNA damage by activating either apo- one main function seems to be its ability to activate 42 transcription of genes containing a p53 response 43 element. The transcription domain is localized in the 44 amino-terminal part of the protein (residues 1–42), 45 5 *Corresponding author. Tel.: 133-4-67-613745; fax: 133-4- whereas the DNA binding domain is localized in the 46 6 67-613041. 7 E-mail address: jmportef@valdorel.fnclcc.fr (J.-M. Portefaix). central region (residues 90–290). The carboxy-termi- 47 1 0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. 2 PII: S0022-1759(00)00246-5 3 8660 DTD410 182JUL22000 17:04:39.46 v1.4.154