CAJ | 학술논문

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(MeAb),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2.12～6.70 mg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-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 .
목적 응용항인간암조직중만타라응집소(DSA)강결합적γ-곡안선전이매(GGT)단극륭항체(MeAb),건립기혈청DSA-GGT생물소-련매친화소ELISA검측방법 ,병탐토기재원발성간암(PHC)진단적응용개치.방법 통과McAb기술제비적항DSA-GGT적McAb,채용단백G친화층석주색보순화;순화적McAb경생물소표기후,구건혈청DSA-GGT생물소-련매친화소ELISA검측방법 .용건립적방법 대39례PHC환자화122례비PHC환자진행초보림상응용연구,용P-P개솔도검험119례건강대조혈청DSA-GGT분포상태,의거수시자공작곡선(ROC)학정DSA-GGT진단PHC적cut-off치.결과 획취5주분비McAb잡교류세포제비적복강적액,경순화후McAb단백총량재2.12～6.70 mg지간;McAb적생물소표기솔위48.6%～72.2%.소구건적혈청DSA-GGT생물소-련매친화소ELISA검측방법 적최저검출한위2μg/L;평균비내、비간변이분별위8.9%화11.5%.119빈건강대조혈청DSA-GGT수평정정태분포,x±s위(1.50±0.51)μg/L;경ROC분석학정기진단PHC적림계치위3.25μg/L.39례PHC환자26례DSA-GGT양성,기진단PHC민감도위66.7%;122례비PHC환자10례DSA-GGT양성,기진단특이도위91.8%.결론 소건립적혈청DSA-GGT생물소-련매친화소ELISA검측방법 구유량호적중복성화가고성.림상초보응용표명기진단PHC적민감도、특이도량호,차방법 조작간편,편우림상추엄응용,위PHC실험실진단제공료신방법 .
Objective To prepare monoclonal antibody (McAb) against γ-glutamyhransferase(GGT) firmly bound to datura stramonim (DSA) leetin 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-sepharose 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 inter-assay coefficients of variation were 8.9% and 11.5% respectively. The distribution of DSA-GGT values of 119 healthy subjects showed Gaussian distribution and its Mean ± SD was ( 1.50±0. 51 ) μg/L. Optimal cut-off value (3.25 μg/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.