高尔基蛋白73(GP73)ELISA定量检测方法的建立及临床应用  

Establishment and application of a sandwich ELISA for quantitative measurement of Golgi protein 73

作　　者：周云[1] 包林[2] 徐言[1] 金秋[2] 张黎[2] 张建平[1] 焦永军[2] 

机构地区：[1]南京医科大学第二附属医院普通外科,江苏省210011 [2]卫生部肠道病原微生物重点实验室 江苏省疾病预防控制中心病原微生物研究所

出　　处：《江苏医药》2013年第9期1005-1008,共4页Jiangsu Medical Journal

基　　金：江苏省卫生厅科研项目(H200814);江苏省医学重点人才课题(RC2011082)

摘　　要：目的建立双抗体夹心ELISA定量检测人血清高尔基蛋白73(GP73)的方法,并用于检测血清GP73含量。方法利用杂交瘤法制备纯化的抗GP73单克隆抗体(mAb)进行辣根过氧化物酶(HRP)标记后,通过棋盘滴定法确定包被抗体和酶标抗体及其最适工作浓度;以重组人的GP73抗原为标准品建立标准曲线;以重复性、灵敏性和回收率实验评价ELISA方法。应用该ELISA法对正常人、肝炎、肝硬化及肝癌患者血清中的GP73水平进行定量检测。结果当抗GP73 mAb3D5A10和7H3F5分别为捕获抗体和酶标抗体、工作浓度分别为40μg/ml和1∶4000时,双抗体夹心ELISA法最优。该方法的平均批内和批间变异系数分别为4.6%和7.6%,灵敏度达3.76ng/ml,平均回收率为(102.2±5)%。用本方法重复测定肝癌(33例)、肝硬化(28例)、乙型肝炎(44例)及正常人(44例)血清样本中GP73浓度(中位数)分别为25.9、27.6、16.7、9.9μg/L;除肝癌与肝硬化组间差异无统计学意义外,其余各组间均有统计学差异(P<0.05)。结论成功建立了双抗体夹心ELISA定量检测GP73的方法。检测血清GP73可作为诊断肝脏疾病的重要辅助手段之一。Objective To develop a sandwich ELISA system for quantitative t of human Golgi protein 73 （GP73） and to use it for detecting serum GP73 level in patients. Methods After labeled the purified monoclonal antibody （mAb） with horseradish peroxidase （HRP）, the antibody-pair matching （for capturing and detecting of antigen, respectively） and the optimal concentrations of anti-GP73 mAb were defined by chessboard titration. Standard curve was performed using recombinant human GP73 protein, the effectiveness of this sandwich ELISA system was evaluated by sensitivity, reproducibility and recovery rate. Serum levels of GP73 in healthy people and the patients with HBV positive, liver cirrhosis and hepatocellular carcinoma（HCC） were measured by this system. Results The optimal paired antibodies were anti-GP73 mAb 3D5A10 and HRP labeled anti-GP73 mAb 7H3F5 with optimal concentrations of 40 μg/ml and 1 ：4000, respectively. The sensitivity of this ELISA system was 3.76 ng/ml. The average coefficients of variations were 4. 6% within assay and 7.6% between assays. The recovery rate was （102.2 ±5）%. The GP73 levels in serum of healthy people, HBV positive patients, liver cirrhosis patients, and HCC patients （median） were 9. 9,16.7, 27.6, and 25.9μg/L, respectively. The difference among the groups was significant （P〈0. 05）, except for HCC versus liver cirrhosis. Conclusion A sandwich ELISA system for quantitative measurement of human GP73 has been established successfully, which may be taken as one of the adjuvant markers in the diagnosis of liver diseases.

关 键 词：高尔基蛋白73 原发性肝癌 双抗体夹心ELISA 

分 类 号：R446[医药卫生—诊断学]