血清肝癌特异性γ-谷氨酰转移酶ELISA检测方法的建立及临床应用  

Establishment of ELISA for detection of hepatoma specific γ-glutamyltransferase and its clinical application

作　　者：王念跃[1] 赵伟[2] 刘晨 文剑[1] 张永臣[1] 端木浩[2] 

机构地区：[1]东南大学附属南京第二医院检验科,210003 [2]东南大学附属南京第二医院传染内科,210003 [3]北京晶美基因谷科技有限公司McAb制备中心

出　　处：《中华检验医学杂志》2009年第3期315-320,共6页Chinese Journal of Laboratory Medicine

基　　金：南京市医学重点科技发展项目（ZKX0303）

摘　　要：目的应用抗人肝癌组织中蔓陀罗凝集素（DSA）强结合的γ-谷氨酰转移酶（GGT）单克隆抗体（McAb），建立其血清DSA—GGT生物素-链霉亲和素ELISA检测方法，并探讨其在原发性肝癌（PHC）诊断的应用价值。方法通过McAb技术制备的抗DSA—GGT的McAb，采用蛋白G亲和层析柱色谱纯化；纯化的McAb经生物素标记后，构建血清DSA—GGT生物素-链霉亲和素ELISA检测方法。用建立的方法对39例PHC患者和122例非PHC患者进行初步临床应用研究，用P—P概率图检验119例健康对照血清DSA—GGT分布状态，依据受试者工作曲线（ROC）确定DSA．GGT诊断PHC的cut-off值。结果获取5株分泌McAb杂交瘤细胞制备的腹腔积液，经纯化后McAb蛋白总量在2．12～6．70mg之间；McAb的生物素标记率为48．6％～72．2％。所构建的血清DSA—GGT生物素-链霉亲和素ELISA检测方法的最低检出限为2μg／L；平均批内、批间变异分别为8．9％和11．5％。119份健康对照血清DSA—GGT水平呈正态分布，x±s为（1．50±0．51）μg/L；经ROC分析确定其诊断PHC的临界值为3．25μg／L。39例PHC患者26例DSA—GGT阳性，其诊断PHC敏感度为66．7％；122例非PHC患者10例DSA—GGT阳性，其诊断特异度为91．8％。结论所建立的血清DSA—GGT生物素一链霉亲和素ELISA检测方法具有良好的重复性和可靠性。临床初步应用表明其诊断PHC的敏感度、特异度良好，且方法操作简便，便于临床推广应用，为PHC实验室诊断提供了新方法。Objective To prepare monoclonal antibody （McAb） against γ-glutamyltransferase （GGT） firmly bound to datura stramonim （DSA） lectin from primary hepatic carcinoma （PHC） tissue and establish an avidin-biotin enzyme-linked immunosorbent assay （ELISA） for evaluating the diagnostic value of serum DSA-GGT for PHC. Methods Anti-DSA-GGT monoclonal antibodies were obtained by McAb technology and purified by protein G-sepbarose affinity chromatography. The McAb was labeled with biotin and avidin-biotin ELISA for measurement of serum DSA-GGT was established. Using the avidin-biotin ELISA, serum DSA-GGT levels was detected in 39 patients with PHC, and 122 patients with non-PHC diseases. The distribution of serum DSA-GGT values of 119 healthy subjects were determined by P-P plots. Optimal cut-off value for the diagnosis of PHC was determined by receiver operating characterstic （ROC） curve. Results The protein levels of McAb in the ascites derived from 5 McAb hybridoma cell strains ranged from 2. 12 to 6.70 mg. The biotin-labeled rate varied from 48.6% to 72. 2% respectively. The minimum detection limit of serum DSA-GGT in avidin-biotin ELISA was 2 μg/L. Intra-assay and interassay coefficients of variation were 8.9% and 11.5% respectively. The distribution of DSA-GGT values of 119 healthy subjects showed Ganssian distribution and its Mean ± SD was （ 1.50 ±0. 51 ） μg/L Optimal cutoff value （3.25μg/L） in the diagnosis of PHC was determined by ROC curve. DSA-GGT was positive in 26 out of 39 patients with PHC and 10 out of 122 patients with non-PHC diseases were positive. The sensitivity and specificity of this assay for the diagnosis of PHC were 66. 7% and 91.8% respectively. Conclusions The convenient avidin-biotin ELISA method was successfully established in our laboratory and it showed a good reproducibility and reliability. It may be a potential tool in the diagnosis of PHC to achieve higher sensitivity and specificity.

关 键 词：肝肿瘤 Γ-谷氨酰转移酶 抗体 单克隆 酶联免疫吸附测定 抗生物素蛋白 

分 类 号：R686[医药卫生—骨科学]