【摘要】： 目前,MUC1作為腺癌診斷的腫瘤標志物之一得到廣泛研究。但現(xiàn)有的檢測手段敏感度較低,為提高檢測的敏感度,本科室建立了雙抗體夾心ELISA法。首先,我們制備兩種融合蛋白MUC1-GST和MUC1-MBP,將這兩種蛋白分別免疫家兔和大鼠,獲得兩種抗MUC1多克隆抗體的血清,經(jīng)飽和硫酸銨沉淀、protein A/G純化及抗GST抗體的吸收獲得部分純化的家兔及大鼠抗MUC1多克隆抗體;通過凝血酶溶解MUC1-GST獲得MUC1標準品,經(jīng)過進一步的篩選,確立了雙抗體夾心ELISA法的包被抗體、檢測抗體及二級抗體分別為兔抗MUC1抗體、大鼠抗MUC1抗體和HRP-山羊抗大鼠IgG以及MUC1標準品濃度。采用此方法對健康人和多種腺癌患者的外周血MUC1水平進行檢測。檢測結(jié)果為:120例健康人檢測結(jié)果全為陰性,檢測特異度為100%;乳腺癌檢測敏感度為100%,乳腺良性疾病患者的檢測特異度為78%;胃腸癌檢測敏感度為88.2%,胃腸良性疾病檢測特異度為93%;肺癌檢測敏感度為88.2%,肺良性疾病檢測特異度為100%;子宮和卵巢癌癥檢測敏感度為83%,子宮和卵巢良性疾病檢測特異度為92.5%;肝癌的檢測敏感度為36.4%,肝良性疾病檢測特異度為93%。雙抗體夾心ELISA法與CA15-3試劑盒對乳腺癌患者、乳腺良性疾病患者和健康人群比較檢測結(jié)果,通過繪制ROC曲線對比顯示,雙抗體夾心ELISA法對乳腺癌的診斷準確率明顯高于CA15-3試劑盒。上述研究結(jié)果提示,本研究建立的雙抗體夾心ELISA法對乳腺癌、胃腸癌、肺癌診斷敏感度明顯提高,有望開發(fā)為臨床輔助診斷的常規(guī)試劑盒,尤其有望應(yīng)用于乳腺癌的大規(guī)模篩查及早期診斷。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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