雙抗體夾心ELISA法檢測(cè)腺癌患者外周血MUC1蛋白水平
發(fā)布時(shí)間：2018-11-25 13:58
【摘要】： 目前,MUC1作為腺癌診斷的腫瘤標(biāo)志物之一得到廣泛研究。但現(xiàn)有的檢測(cè)手段敏感度較低,為提高檢測(cè)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(guò)凝血酶溶解MUC1-GST獲得MUC1標(biāo)準(zhǔn)品,經(jīng)過(guò)進(jìn)一步的篩選,確立了雙抗體夾心ELISA法的包被抗體、檢測(cè)抗體及二級(jí)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(biāo)準(zhǔn)品濃度。采用此方法對(duì)健康人和多種腺癌患者的外周血MUC1水平進(jìn)行檢測(cè)。檢測(cè)結(jié)果為:120例健康人檢測(cè)結(jié)果全為陰性,檢測(cè)特異度為100%;乳腺癌檢測(cè)敏感度為100%,乳腺良性疾病患者的檢測(cè)特異度為78%;胃腸癌檢測(cè)敏感度為88.2%,胃腸良性疾病檢測(cè)特異度為93%;肺癌檢測(cè)敏感度為88.2%,肺良性疾病檢測(cè)特異度為100%;子宮和卵巢癌癥檢測(cè)敏感度為83%,子宮和卵巢良性疾病檢測(cè)特異度為92.5%;肝癌的檢測(cè)敏感度為36.4%,肝良性疾病檢測(cè)特異度為93%。雙抗體夾心ELISA法與CA15-3試劑盒對(duì)乳腺癌患者、乳腺良性疾病患者和健康人群比較檢測(cè)結(jié)果,通過(guò)繪制ROC曲線對(duì)比顯示,雙抗體夾心ELISA法對(duì)乳腺癌的診斷準(zhǔn)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(duì)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(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2356361
[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(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 丁昌平,柳健,陳新華;TSGF、CA_(125)、SF、CEA聯(lián)檢對(duì)卵巢癌診斷價(jià)值的探討[J];放射免疫學(xué)雜志;2001年01期
2 胡家露，李新華;胃腸道惡性腫瘤的實(shí)驗(yàn)室檢查[J];中國(guó)實(shí)用內(nèi)科雜志;1996年01期
3 高海燕,朱華,叢占杰;三種肝癌標(biāo)志物對(duì)肝癌診斷特異性的評(píng)價(jià)[J];實(shí)用腫瘤學(xué)雜志;2004年05期
4 曾波航,蔡燕燕,呂嘉春,鄔勇堅(jiān),江素華,于憲;小細(xì)胞肺癌腫瘤標(biāo)志物:血清NSE與CYFRA21-1和CEA[J];現(xiàn)代臨床醫(yī)學(xué)生物工程學(xué)雜志;2003年03期
5 徐宏勇;徐立;高建宏;楊建軍;李開宗;竇科峰;;抗癌胚抗原單鏈抗體的原核表達(dá)及對(duì)人胃癌的檢測(cè)[J];世界華人消化雜志;2006年18期
6 孫巍,王瓊,劉娟,張春榮,高學(xué)碩,萬(wàn)文徽;血清腫瘤標(biāo)志在結(jié)直腸癌診斷中的應(yīng)用[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2000年01期
7 田艷濤,李樹玲,傅西林;乳腺乳頭狀瘤病及其癌變的全乳腺切片病理組織學(xué)研究[J];中華腫瘤雜志;1997年04期
相關(guān)碩士學(xué)位論文 前1條
1 馬吉春;重組MUC1融合蛋白的制備及檢測(cè)MUC1雙抗體夾心ELISA方法的建立[D];吉林大學(xué);2007年
，本文編號(hào)：2356361
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2356361.html
最近更新
教材專著
