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.

Role of superoxide dismutase in hepatitis B virus-related hepatocellular carcinoma

“…[28] Therefore, an additional marker, such as SOD, has been suggested to be simultaneously measured to improve the HCC detection rate. This study showed a significant increase in serum AFP in HCC patients compared to HBV patients without HCC and healthy controls.…”

A review of GOLPH2, an oncogenic protein and novel therapeutic options for GOLPH2 driven tumours

“…Techniques of GOLPH2 quantification have evolved in recent years from time-consuming Western Blotting (WB) [11]. via Enzyme linked Immuno Sorband Assays (ELISA) [11][12][13][14][15] to particle based unpretentious bioassays. Quantification of GOLPH2 expression in tumour tissue by immunohistochemistry (IHC), a routine clinical technique, paved the way for exploration of GOLPH2 expression in solid tumours like HCC, prostate [16] and gastric cancer [17], lung adeno carcinoma [18,19], glioblastoma [20] and melanoma [21].…”

A review on the diagnosis and treatment of hepatocellular carcinoma with a focus on the role of Wnts and the dickkopf family of Wnt inhibitors

“…Ju et al found that a type II Golgi-specific membrane protein, Golgi phosphoprotein 2, was overexpressed in HCC patients and it was more sensitive for diagnosis of HCC (76%) than the traditional biomarker AFP (70%). 11 Therefore, Golgi phosphoprotein 2 may be a new and effective serum biomarker for HCC diagnosis. Youns et at described that talin-1 has the potential to serve as an important prognostic marker for the diagnosis of HCC, with superior sensitivity and specificity when compared to AFP.…”