目的 建立可用于血清蔓陀罗凝集素(DSA)-γ-谷氨酰基转肽酶(GGT)检测的酶联免疫吸附法(ELISA),并探讨其在原发性肝癌(PHC)诊断中应用价值.方法 制备抗DSA-GGT 单克隆抗体(McAb),经蛋白G亲和层析柱色谱纯化后进行生物素标记,构建血清DSA-GGT生物素-链霉亲和素ELISA检测方法.用建立的方法对健康者、PHC患者和非PHC患者进行DSA-GGT检测,并进行方法学评价;采用受试者工作曲线(ROC曲线)确定DSA -GGT诊断PHC的临界值.结果 所构建ELISA检测方法的最低检测限为2 μg/L,平均批内、批间变异分别为8.9%和11.5%.健康者血清DSA-GGT水平呈正态分布,参考范围为(1.50±0.51)μg/L;经ROC曲线分析,确定其诊断PHC的临界值为3.25 μg/L;其诊断PHC灵敏度为66.7%(28/40)、特异度为91.8%(112/122).结论 所建立的血清DSA-GGT生物素-链霉亲和素ELISA检测方法具有良好方法学性能,其诊断PHC的灵敏度、特异度良好,为PHC实验室诊断提供了新的方法.%Objective To establish an enzyme-linked immunosorbent assay (ELISA) for the detection of datura stramonim (DSA)-'y-glutamyltransferase(GGT) and investigate its clinical significance for the diagnosis of primary hepatic carcinoma (PHC) . Methods Anti-DSA-GGT monoclonal antibodies(McAb) was prepared and labeled with biotin,after being purified by protein G-sepharose affinity chromatography .And avidin-biotin ELISA for measurement of serum DSA-GGT was established and evaluated by detecting serum DSA-GGT in healthy subjects,patients with or without PHC .Cut-off value of DSA-GGT for the diagnosis of PHC was determined by receiver operating characteristic curve (ROC curve) .Results The minimum detection limit of serum DSA-GGT by avidin-biotin ELISA was 2μg/L .Intra- and inter-assay coefficients of variation were 8 .9/0 and 11 .5% respectively .The distribution of DSA-GGT values showed Gaussian distribution and the reference range was (1 .50zb0 .51)μg/L .Optimal cut-off value was 3 .25μg/L for the diagnosis of PHC,determined by ROC curve,with diagnostic sensitivity and specificity of 66 .7% (28/40) and 91.8% (112/122),respectively .Conclusion The convenient avidin-biotin ELISA method was successfully established,with fine re-pro ducibility and reliability .It might be a potential tool for the diagnosis of PHC to achieve higher sensitivity and specificity .
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