结缔组织生长因子elisa定量检测方法的建立及初步应用
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卫生部科学研究基金资助(wkj2004-2-12);南京医科大学肿瘤中心招标课题(08zlkf06)


establishment and application of a sandwich elisa for quantitative measurement of human connective tissue growth factor
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    摘要:

    目的:建立定量检测人结缔组织生长因子(ctgf)双抗体夹心elisa方法,并应用此方法检测人血清中ctgf含量-方法:利用简易过碘酸钠法对纯化的抗ctgf单克隆抗体(mabs)进行hrp标记后,行抗体配对实验;通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度;以纯化的ctgf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-用该方法初步对正常人-hbsag阳性非肝癌患者及hbsag阳性肝癌患者血清中的ctgf水平进行检测-结果:最佳配对组合为抗ctgf mab 3h5和hrp标记的抗ctgf mab 4c5,最适工作浓度分别为40 μg/ml 和1/4 000,该方法的平均批内-批间变异系数分别为4.59%和5.70%,灵敏度达1.46 ng/ml,平均回收率为(98.73±4.84)%-用本方法测定35例hbsag阳性肝癌患者-76例hbsag阳性非肝癌患者和40例正常人血清ctgf水平,与正常对照组[2.65(2.16~3.18) ng/ml]比较,hbsag阳性非肝癌组[5.53(2.74~8.23) ng/ml] 和hbsag阳性肝癌组[8.27(4.81±13.65) ng/ml] 血清ctgf水平均显著升高(p < 0.001);且hbsag阳性肝癌组与hbsag阳性非肝癌组比较也明显升高(p=0.014)-结论:建立了一种可用于检测人ctgf的双抗体夹心elisa方法-初步应用结果表明ctgf水平在hbsag阳性肝癌患者血清中较hbsag阳性非肝癌患者-正常人异常升高,提示ctgf分子有望成为hbsag阳性肝癌早期预警标志物-

    Abstract:

    objective:to develop a sandwich elisa system for quantitative measurement of the human connective tissue growth factor(ctgf), and to detect the sera ctgf level of human. methods:after labeled the purified monoclonal antibodies(mabs) with horseradish peroxidase (hrp) by sodium oxidation method, the antibody-pair matching(for capturing and detection of antigen, respectively) was performed using anti-ctgf mab as coating antibody and hrp labeled anti-ctgf mab as labeled antibody,the optimal concentrations of anti-ctgf mabs were defined by titration. standard curve was performed using purified ctgf protein,the effectiveness of this sandwich elisa system was evaluated by sensitivity, reproducibility and recovery rate. the ctgf levels in sera of health people, hbsag(+) without hepatocellular carcinoma (hcc) patients and hbsag(+) with hcc patients were measured by this system. results:the optimal paired antibodies were anti-ctgf mab 3h5 and hrp labeled anti-ctgf mab 4c5 with optimal concentrations of 40 μg/ml and 1∶4 000 respectively. the sensitivity of this elisa system was 1.46 ng/ml. the average coefficients of variations were 4.59% within assay and 5.70% between assays. the recovery rate was (98.73±4.84)%. the ctgf levels in sera of 40 health people, 76 hbsag(+) without hcc patients and 35 hbsag(+) with hcc patients were [2.65(2.16±3.18) ng/ml], [5.53(2.74±8.23)ng/ml],[8.77(4.81±13.65) ng/ml],respectively. the difference among these groups showed great significance(p < 0.001). conclusion:a sandwich elisa system for quantitative measurement of human ctgf was successfully established. the serum level of ctgf in hbsag(+) with hcc patients raises significantly compare with hbsag(+) without hcc patients and normal group. this suggests that ctgf may be a potential biomarker for the forewarning of hbsag (+) with hcc.

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詹 峰,曾晓燕,张 晓,徐 言,张建平,焦永军.结缔组织生长因子elisa定量检测方法的建立及初步应用[J].南京医科大学学报(自然科学版),2011,(4):449-453

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  • 收稿日期:2010-10-19
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