Portefaix J M, Thebault S, Bourgain-Guglielmetti F, Del Rio M, Granier C, Mani J C, Navarro-Teulon I, Nicolas M, Soussi T, Pau B
CNRS UMR5094, CRLC Val d'Aurelle/Bât Recherche, Rue de la Croix Verte, 34298 Cedex 5, Montpellier, France.
J Immunol Methods. 2000 Oct 20;244(1-2):17-28. doi: 10.1016/s0022-1759(00)00246-5.
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.
这项工作的目的是研究针对p53蛋白N端的抗体的反应性。首先，我们分析了来自人、小鼠和兔血清的抗p53抗体与源自人、小鼠和非洲爪蟾p53的肽段的交叉反应性。接下来，我们更精确地表征了一系列针对p53 N端部分的单克隆抗体，这些抗体是通过用全长人p53或非洲爪蟾p53免疫小鼠产生的。对于这些单克隆抗体中的每一种，我们通过多聚肽合成的斑点法在人p53上定位了所识别的表位，通过丙氨酸扫描置换实验确定了p53中参与相互作用的关键残基，并使用实时相互作用分析确定了动力学参数。根据它们的表位和动力学特征以及与鼠p53的交叉反应性，这些抗体可分为两组。我们的结果表明，其中一些单克隆抗体与p53相互作用所涉及的关键残基对应于p53与mdm2蛋白相互作用所涉及的关键残基。因此，这些抗体可能是研究p53调控的有力工具。
