title: Preparation and characterization of anti-GP73 monoclonal antibodies and development of double-antibody sandwich ELISA.
abstract: BACKGROUND
  Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variables representing tumor growth and invasiveness. The aim of this study was to prepare a pair of monoclonal antibodys (mAbs) against GP73 and develop a newly designed double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis of HCC.
  
  
  METHODS
  Produced by prokaryotic expression, the purified recombinant GP73 (rGP73), produced by prokaryotic expression, was used to immunize the Balb/c mice. Two hybridoma cell lines against GP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titers of anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealed that anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully established and validated by 119 HCC and 103 normal serum samples.
  
  
  RESULTS
  showed that the detection limit of this method could reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those of in healthy controls (mean=70.92 ng/ml).
  
  
  CONCLUSIONS
  Results of our study not only showed that sGP73 levels of HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratory homemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide a solid foundation for subsequent clinical applications.
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing, China E-mail : sijin_efy@njmu.edu.cn.
authors.forename: Qi-Wen
authors.initials: QW
authors.lastname: Li
authors.forename: Hong-Bing
authors.initials: HB
authors.lastname: Chen
authors.forename: Zhi-Yang
authors.initials: ZY
authors.lastname: Li
authors.forename: Peng
authors.initials: P
authors.lastname: Shen
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Qu
authors.forename: Lai-Ling
authors.initials: LL
authors.lastname: Gong
authors.forename: Hong-Pan
authors.initials: HP
authors.lastname: Xu
authors.forename: Lu
authors.initials: L
authors.lastname: Pang
authors.forename: Jin
authors.initials: J
authors.lastname: Si
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000097:blood; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000175:diagnosis*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D056945:Hep G2 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008113:Liver Neoplasms / Q000097:blood / Q000175:diagnosis*; D008565:Membrane Proteins / Q000097:blood / Q000276:immunology*; D008807:Mice, Inbred BALB C
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D008565:Membrane Proteins