title: A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues.
abstract: BACKGROUND
  The ras genes play an important role in the development and progression of human tumours. Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling. In this study, we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours.
  
  
  METHODS
  The coding sequences of H-ras, K-ras, and N-ras were separately ligated into the vector pET-28a(+). Then, recombinant expressing plasmids were induced by IPTG for p21Ras expression in E. coli. Hybridoma cell lines producing anti-p21Ras monoclonal antibodies were isolated using wildtype p21Ras proteins as immunogens. Anti-p21Ras scFv antibody was prepared from the hybridoma by the phage scFv display method. The immunoreactivity of the anti-p21Ras monoclonal antibody and the scFv antibody was identified by ELISA and immunocytochemistry.
  
  
  RESULTS
  We prokaryotically expressed wildtype H-p21Ras, K-p21Ras and N-p21Ras and generated the hybridoma cell line KGH-R1, producing anti-p21Ras monoclonal antibodies. It was demonstrated that KGH-R1 monoclonal antibody could recognize wildtype and mutated H-p21Ras, K-p21Ras and N-p21Ras in human tumour cell lines. In all 14 types of primary human cancer tissues tested, the monoclonal antibody presented strong immunoreactivity but showed weak or negative immunoreactivity in the corresponding normal tissues. Subsequently, we prepared anti-p21Ras scFv from hybridoma KGH-R1, which showed the same immunoreactivity as the original monoclonal antibody. Sequence analysis demonstrated that the nucleotides and amino acids of the scFv exhibited an approximately 50 % difference from the anti-p21Ras scFv reported previously.
  
  
  CONCLUSIONS
  This study presents a novel anti-p21Ras scFv antibody. Our data suggest that the scFv may be useful for ras signalling blockage and may be a potential therapeutic antibody for ras-derived tumours.
authors.affiliation: Department of Pathology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China. yangjulun@sina.com.
authors.forename: Ju-Lun
authors.initials: JL
authors.lastname: Yang
authors.affiliation: Department of Molecular Biology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Du-Xian
authors.initials: DX
authors.lastname: Liu
authors.affiliation: Department of Molecular Biology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Shi-Jian
authors.initials: SJ
authors.lastname: Zhen
authors.affiliation: Department of Molecular Biology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Yun-Gang
authors.initials: YG
authors.lastname: Zhou
authors.affiliation: Department of Pathology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Dai-Jun
authors.initials: DJ
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Li-Ying
authors.initials: LY
authors.lastname: Yang
authors.affiliation: Department of Pathology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Hao-Bing
authors.initials: HB
authors.lastname: Chen
authors.affiliation: Department of Pathology, Kunming General Hospital/Kunming Medical University, Kunming, 650032, Yunnan Province, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Feng
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000494:pharmacology; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D045325:HCT116 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D006825:Hybridomas / Q000166:cytology; D061986:MCF-7 Cells; D051379:Mice; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)