双抗体夹心ELISA法检测腺癌患者外周血MUC1蛋白水平

发布时间：2018-11-25 13:58

【摘要】： 目前,MUC1作为腺癌诊断的肿瘤标志物之一得到广泛研究。但现有的检测手段敏感度较低,为提高检测的敏感度,本科室建立了双抗体夹心ELISA法。首先,我们制备两种融合蛋白MUC1-GST和MUC1-MBP,将这两种蛋白分别免疫家兔和大鼠,获得两种抗MUC1多克隆抗体的血清,经饱和硫酸铵沉淀、protein A/G纯化及抗GST抗体的吸收获得部分纯化的家兔及大鼠抗MUC1多克隆抗体;通过凝血酶溶解MUC1-GST获得MUC1标准品,经过进一步的筛选,确立了双抗体夹心ELISA法的包被抗体、检测抗体及二级抗体分别为兔抗MUC1抗体、大鼠抗MUC1抗体和HRP-山羊抗大鼠IgG以及MUC1标准品浓度。采用此方法对健康人和多种腺癌患者的外周血MUC1水平进行检测。检测结果为:120例健康人检测结果全为阴性,检测特异度为100%;乳腺癌检测敏感度为100%,乳腺良性疾病患者的检测特异度为78%;胃肠癌检测敏感度为88.2%,胃肠良性疾病检测特异度为93%;肺癌检测敏感度为88.2%,肺良性疾病检测特异度为100%;子宫和卵巢癌症检测敏感度为83%,子宫和卵巢良性疾病检测特异度为92.5%;肝癌的检测敏感度为36.4%,肝良性疾病检测特异度为93%。双抗体夹心ELISA法与CA15-3试剂盒对乳腺癌患者、乳腺良性疾病患者和健康人群比较检测结果,通过绘制ROC曲线对比显示,双抗体夹心ELISA法对乳腺癌的诊断准确率明显高于CA15-3试剂盒。上述研究结果提示,本研究建立的双抗体夹心ELISA法对乳腺癌、胃肠癌、肺癌诊断敏感度明显提高,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。
[Abstract]:At present, MUC1 as one of the tumor markers in the diagnosis of adenocarcinoma has been widely studied. But the sensitivity of the existing detection methods is low. In order to improve the sensitivity, a double antibody sandwich ELISA method was established. First, we prepared two fusion proteins, MUC1-GST and MUC1-MBP, which immunized rabbits and rats respectively, and obtained two kinds of sera against MUC1 polyclonal antibodies, which were precipitated by saturated ammonium sulfate. The polyclonal antibodies against protein A / G and anti GST antibodies were partially purified from rabbits and rats. The standard MUC1 standard was obtained by thrombin dissolution of MUC1-GST. After further screening, the coated antibody of double antibody sandwich ELISA method was established. The detection antibody and the secondary antibody were rabbit anti-MUC1 antibody, respectively. Rat anti MUC1 antibody and HRP- goat anti rat IgG and MUC1 standard concentration. The level of MUC1 in peripheral blood of healthy people and various adenocarcinoma patients was detected by this method. The results were as follows: all the 120 healthy people were negative, the detection specificity was 100, the sensitivity of breast cancer was 100, the detection specificity of benign breast disease was 78. The sensitivity of gastrointestinal cancer was 88.2, the specificity of benign gastrointestinal diseases was 933. The sensitivity of lung cancer was 88.2and the specificity of benign diseases was 100. The sensitivity of cancer detection of uterus and ovary was 83.The specificity of benign diseases of uterus and ovary was 92.5, the sensitivity of liver cancer was 36.4 and the specificity of benign diseases of liver was 933. The results of double antibody sandwich ELISA assay and CA15-3 kit were compared with those of breast cancer patients, benign breast disease patients and healthy people. The ROC curves were compared with each other. The diagnostic accuracy of double antibody sandwich ELISA for breast cancer was significantly higher than that of CA15-3 kit. These results suggest that the diagnostic sensitivity of the double antibody sandwich ELISA assay for breast cancer, gastrointestinal cancer and lung cancer is significantly improved, and it is expected to be developed as a routine kit for clinical diagnosis. Especially, it is expected to be used in large-scale screening and early diagnosis of breast cancer.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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