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.
Highlights
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high malignancy and mortality due to lack of early diagnosis and its resistance to conventional chemotherapy [1]
The average serum content of Golgi phosphoprotein 2 (Golph2) in HCC patients is >100 μg/l, consistent with other studies using sandwich enzyme-linked immunosorbent assay (s-ELISA) [8]. Since it was first identified in acute giant-cell hepatitis, tissue-based Golph2 overexpression has been confirmed in HCC [8,14], lung adenocarcinoma [15], seminomas [16], renal cell cancer [17] and other malignant diseases
A large number of studies have indicated that the serum level of Golph2 may be a more reliable biomarker for the early diagnosis of HCC than AFP
Summary
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high malignancy and mortality due to lack of early diagnosis and its resistance to conventional chemotherapy [1]. HCC affects approximately one million people every year worldwide, with the incidence equal to the mortality. In 2008, HCC was listed as the third most common cause of cancer-related mortality [2]. Early diagnosis is crucial in order to increase the survival rate for patients [3]. Alpha‐fetoprotein (AFP) together with imaging and pathology detection are commonly used in early clinical diagnosis of liver cancer. The specificity and sensitivity of AFP for liver cancer screening are not satisfactory. With the development of molecular biology, a number of new types of tumor markers have been discovered
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