title: Monoclonal antibody preparation of Golgi phosphoprotein 2 and preliminary application in the early diagnosis of hepatocellular carcinoma.
abstract: Golgi phosphoprotein 2 (Golph2) is a type II Golgi‑specific membrane protein, which has been found to be overexpressed in hepatocellular carcinoma (HCC) patients. The sensitivity of diagnosis of HCC using Golph2 (76%) was markedly elevated compared with alpha‑fetoprotein (AFP) (70%), and Golph2 is expected to be a novel and effective serum biomarker for the diagnosis of HCC. The aim of this study was to prepare monoclonal antibodies against Golph2 and to establish double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which will be used in diagnostics, therapeutics and as a tool in understanding the role of Golph2 in the pathogenesis of liver diseases and cancer. In this study, fusion protein TRX-Golph2 was expressed and purified using an Escherichia coli system. BALB/c mice were immunized with TRX-Golph2 recombinant protein. The hybridoma technique was used for the production of anti-Golph2 monoclonal antibody. Hybridoma clones were screened using indirect ELISA and anti-Golph2 monoclonal antibody was produced in the ascites of BALB/c mice. The specificity of anti-Golph2 monoclonal antibody was detected by western blot analysis and immunocytochemistry. s-ELISA was established using horseradish peroxidase (HRP)‑labeled anti-Golph2 monoclonal antibody and used to detect the antigen in the serum of HCC patients. As a result, five stable hybridoma cell clones (5C6D5, 5B7F5, 7F5F3, 8A7B4, 8C9E8) producing anti-Golph2 monoclonal antibody were established. The highest titer of anti-Golph2 monoclonal antibody (5C6D5) was 1:51,200. Western blot analysis revealed that anti-Golph2 monoclonal antibody had a high specificity for Golph2 protein. Anti-Golph2 monoclonal antibody was HRP-labeled and the optimal working concentration was found to be 1:500. The levels of antigen in a proportion of HCC patients were shown to be significantly higher compared to those found in healthy controls.
authors.affiliation: Cancer Research Institute, Key Laboratory of Carcinogenesis and Cancer Invasion Ministry of Education, Key Laboratory of Carcinogenesis Ministry of Health, Central South University, Changsha, Hunan 410078, P.R. China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ju
authors.forename: Yanjie
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Liu
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Pingli
authors.initials: P
authors.lastname: Xie
authors.forename: Yuehui
authors.initials: Y
authors.lastname: Li
authors.forename: Guan-Cheng
authors.initials: GC
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000175:diagnosis* / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; C410824:GOLM1 protein, human; D008565:Membrane Proteins