Preparation And Application Of Monoclonal Antibody Of Human Golgi Protein73

ObjectionHepatocellular carcinoma (HCC) is one of the most frequent malignant tumorsand is the second most common cause of cancer death in China. The incidence andmortality of HCC is found increasing year by year. The worldwide new cases of HCCare approximately560,000each year, of which nearly50%of cases occur in China.Surgical resection remains the main treatment of HCC. But5-year survival is only30%to40%for the patients who accepted radical surgery. The prognosis should besignificantly improved by early diagnosis and treatment. HCC conventional screeningand diagnosis methods include physical examinations, imaging technologies andblood serum Alpha fetoprotein (AFP) examination.Nowadays, the diagnosis of HCCmainly due to AFP. It shows a sensitivity of50%to70%, but only40%for early HCC.Early diagnosis of HCC urgently needs to be improved. Thus, hunting for a moresensitive and specific serum biomarker to establish a HCC early warning systemis desired. Recent studies revealed that the sensitivity and specificity of GP73in sera aresuperior to AFP in the diagnosis of HCC. GP73is expected to be a potential biomarker for earlydiagnosis of HCC. So, the aim of this research is to prepare the monoclonal antibody(mAb) against GP73and identify the biological characteristics of the mAb. To develop aSandwich ELISA method for quantitative measurement of GP73, and to detect the sera GP73level of human.Methods1. RT-PCR technology was used to amplify the sequence of gp73.The expression vector pComb3XSS-gp73was constructed and transformed into E.coli. Top10F′forexpression by IPTG.The fusion protein was identification by Western blot andpurified with their fusion partner by His TrapFF affinity chromatography.2. The purified His-GP73fusion protein was used to immunize the BALB/cmice. Then, the mouse myeloma cells (Sp2/0) were fused with spleen cells fromBALB/c immunized by the purified protein. Subsequently, three times limiteddilution method was used to screen hybridoma cell lines.3. The titer of the mAb was detected by ELISA and its specificity was analyzedby Western blot. The subclasses of mAb were identified by the kit for MouseMonoclonal subclasses. The sera level of GP73in HCC patients and healthy peoplewas measured by co-immunoprecipitation(IP).4. a-GP73mAb3D5A10was designed as the capturing of antibody and thea-GP73mAb5E2G6-HRP as the detection of antibody, which optimal concentrationswere defined by titration. Standard curve was performed using purified GP73protein.5. A sandwich ELISA assay was performed to measure the GP73level of sera inhealthy people, chronic hepatitis and HCC patients.Results1. The expression vector pComb3XSS-gp73was constructed successfully.2. The GP73fusion protein was successfully expressed and purified. Theconcentration and purity of the protein is2.5mg/mL and more than80%respectively.3. Five hybridoma cell lines against GP73were obtained, which named3D5A10,8C4G5,5E2G6,7C8F4and7H3F5. The heavy chain of immunoglobulin subclasses wasIgG1, IgG1, IgG2a, IgG2a, IgG3respectively. The light chains were all κ. And thetitration is above1:160,000. Western blot showed that GP73mA can identify thenature GP73protein in the lysate of cells. IP revealed that the mAb could combinewith GP73in human sera with high specificity. The level of GP73in HCC patients is much higher than that of healthy people.4. A sandwich ELISA assay for quantitative measurement human GP73wassuccessfully established and the sensitivity of this assay was1.56ng/mL.5. The GP73level of sera in66healthy people,28cirrhosis and33HCC patientswere quantitative measured by using the established sandwich ELISA assay. The levelof GP73in HCC patients and chronic hepatitis patients were higher than that inhealthy people (P<0.05). The level of GP73in HCC patients was also higher thanthat in chronic hepatitis patients (P<0.05).ConclusionsIn our research, the expression vector pComb3XSS-gp73was constructedsuccessfully.GP73fusion protein was used as antigen and we successfully preparea-GP73mAb. The GP73level of sera in healthy people, chronic hepatitis and HCCpatients were quantitative measured by using the established sandwich ELISA assay.The level of GP73in HCC patients was higher than that in healthy individuals andchronic hepatitis patients. All the results demonstrated that GP73may be a potentialbiomarker for the diagnosis of HCC.