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Volume 159, Issue 3 p. 529-534
Free Access

Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression

Lawrence BANKS

Corresponding Author

Lawrence BANKS

Laboratory of Molecular Virology, Imperial Cancer Research Fund, London

Correspondence to L. Banks, Laboratory of Molecular Virology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, England WC2A 3PXSearch for more papers by this author
Greg MATLASHEWSKI

Greg MATLASHEWSKI

Laboratory of Molecular Virology, Imperial Cancer Research Fund, London

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Lionel CRAWFORD

Lionel CRAWFORD

Laboratory of Molecular Virology, Imperial Cancer Research Fund, London

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First published: September 1986
Citations: 323

Abstract

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-1 cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with β-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognised by the antibodies. At least two previously unrecognised epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-1) cells cause expression of human p53 recognised by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.

Abbreviations

  • ELISA
  • enzyme-linked immunoassay
  • NP40
  • Nondidet P40
  • SDS-PAGE
  • sodium dodecyl sulphate/polyacrylamide gel electrophoresis
  • bp
  • base pairs
  • SV40
  • simian virus 40