Gp73 Monoclonal Antibody Diagnosis Of Liver Cancer

Objective U.S. scientists found that the Golgi protein 73(GP73) may be a better serum marker for diagnosis of liver cancer in recent years. Although GP73 polyclonal antibodies are widely used in detection of serum GP73, the poor reproducibility and scale of their production,and limiting their clinical application. In this study, we tested the sensitivity and specificity of GP73 monoclonal antibody for the diagnosis of HCC. Methods Large-scale detection of clinical HCC patients to expression of serum GP73 level, we must first prepare GP73 monoclonal antibodies.In this study, using the GP73 antigen (4μg/ml) to immune BALB/c female mice with age of 6 to 8 weeks, after the basic immunization and booster immunization, blood samples were obtained in mice to test antibody titers. Spleen cells from immunized mouse with SP2/0 cells induced fusion in 50 PEG. After repeated screening hybridoma cells and cloning of hybridoma cells, then BALB/c female mice were inoculated intraperitoneally with hybridoma cells, gathered ascites after 1 week, centrifuged and collected the supernatant which using protein A affinity chromatography purified. Tested antibody protein solution value with UV spectrophotometer, using the formula C (mg/ml protein)= 3 to calculate the antibody concentration. To investigate the sensitivity and specificity of GP73 monoclonal antibody for the diagnosis of HCC, Western Blotting was used for determination of human serum relative content of protein in GP73 in this study.Electrophoresis of serum to be detected, we used iBlot rapid instrument to transfer film. Milk or BSA closed, placed in hybridization membrane, added primary anti-body and incubated in 4℃shaker.TBST eluted, then incubated at room temperature after added secondary anti-body, and TBST eluted, used the ECL in darkroom for exposure. We scanned X-ray and analyzed the gray value of each band. Results Using monoclonal antibody to test the levels of normal control (1.3±0.1)RU, and the serum levels of liver cancer compared with normal control were significantly higher (5.8±0.8)RU vs.(1.3±0.1)RU, P< 0.001, and serum GP73 levels of HCC (polyclonal antibody) compared with normal control were significantly higher (7.8±0.7)RU vs.(1.1±0.03)RU, P<0.001. The sensitivity and specificity of GP73 monoclonal antibody for diagnosis of HCC were 84.7% and 93.5%, and GP73 polyclonal antibody were 78% and 93.5%, respectively, while the sensitivity and specificity for diagnosis of HCC using AFP were 67.8% and 74.2%.The odds ratio (OR) of GP73 monoclonal antibody is 7.18 in logistic regression analysis, and the OR of GP73 polyclonal antibody is 1.51. Conclusions Monoclonal antibodies can be detected in serum GP73 protein levels in patients with liver cancer, has a higher sensitivity and specificity than AFP, may also be superior to current detection with GP73 polyclonal antibody.