Monoclonal Antibody Preparation Of Golgi Phosphoprotein2and Preliminary Application Of The Early Diagnosis Of Hepatocellular Carcinoma

Background and Objectives:Liver cancer is currently one of the malignant tumors seriously influencing human health, and its incidence ranks fifth among all malignant tumors half of which is from China. It has been a major killer which severely threatens people’s health in our country. The early symptoms of liver cancer are not obvious, which is its characteristics, together with unsatisfactory biomarkers for early diagnosis leading to be late in the time of diagnosed, seriously reducing the effects of treatment. In recent studies, the golgi membrane protein Ⅱ (Golph2) had been preliminarily confirmed that it has the characteristics of biomarkers for early diagnosis of liver cancer, and is expected to become a new generation biomarker which maybe take the place of AFP. Therefore, this research aims to set up a method and technology which be quick and efficient in detection of Golph2content in serum, and can lay a foundation for early diagnosis of liver cancer patients.Methods:Prokaryotic Golph2expression system be built and the fusion protein TRX-Golph2was induced and expressed by IPTG. The soluble fusion protein was purified in the native condition by His-tag magnetic bead purification kit and identified by Western blot. The mice BALB/c were immunized with the purified protein TRX-Golph2. Anti-Golph2monoclonal antibodies were developed by hybridoma technique. Their specificity and relative affinity were analyzed by Western blot, immunocytochemistry and ELISA. Anti-Golph2mAbs were purified by Sepharcryl S-300HR column chromatography and labeled with HRP(horse radish peroxidase) through the means of modified sodium periodate oxidation method. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established and optimized through the means of Chessboard titration method and was applied to detect the level of Golph2in serum.Results:1. The recombinant plasmid pET21a(+)-TRX-Golph2was transformed into E.coli RosettaTM and optimal expression of recombinant proteins was achieved through properly controlling the concentration of Isopropyl-b-D-thiogalactopyranoside(IPTG,1.0mM) and growth conditions(30℃,10h). The protein was identified by Western blot analysis using anti-his-tag antibody. Analysis of SDS-PAGE identified that about one-third of the total amount of interest protein was soluble.2. Based on the affinity between His-tag sequence and Ni+, the interest fusion protein was successfully purified in native condition by affinity chromatography.3. BALB/c mice were immunized with purified fusion protein TRX-Golph2by peritoneal injection. After meeting requirement, the hybridoma cells were successfully prepared, and five hybridoma cell lines (5C6D5,5B7F5,7F5F3,8A7B4,8C9E8) that stably secreted anti-Golph2MAb were obtained, four of which were IgM and one was IgG1via identification of subtype antibody kits. Titer of ascitic fluid were above1:10000by the indirect ELISA analysis. The anti-Golph2mAbs had good specificity by Western blot and immunocytochemistry analysis.4. Anti-Golph2mAbs were successful purified by Sepharcryl S-300HR column chromatography and labeled with HRP through the means of modified sodium periodate oxidation method. By direct ELISA, the working concentration of labeled monoclonal antibody is1:500.Double antibody sandwich ELISA was established and optimized through the means of Chessboard titration method.The best working concentration of HRP-labeled MAb and the dilution ratio of serum were fixed on1:500and1:2respectively by Chessboard titration method. 5. The levels of antigen in samples were detected by Double antibody sandwich ELISA. The result showed that the median serum level of Golph2in the HCC group exceeded significantly that in healthy controls, and the differences were considered statistically significant (p<0.05).Conclusions:1. The fusion protein TRX-Golph2was successfully induced, expressed and purified in the native condition.2. The anti-Golph2mAbs with both high sensitivity and high specificity were successfully prepared.3. Anti-Golph2mAbs were successful purified and labeled with HRP. The working concentration of labeled monoclonal antibody is1:500.4. Double antibody sandwich ELISA in order to detect the level of Golph2in serum was successfully established and optimized via Chessboard titration method. The s-ELISA can be used for detecting the level of Golph2in serum.5. Compared with the control group, preliminary research confirmed that the level of Golph2in serum of HCC overexpressed.