Affimer-抗體雜合化學(xué)發(fā)光免疫分析法的建立及其在血清磷脂酰肌醇蛋白聚糖-3檢測(cè)中的應(yīng)用研究
發(fā)布時(shí)間：2019-06-20 02:03
【摘要】：磷脂酰肌醇蛋白聚糖-3(glypican-3,GPC3)是一個(gè)有價(jià)值的肝細(xì)胞癌(HCC)診斷標(biāo)志物,然而到目前為止,尚未有可靠的血清學(xué)檢測(cè)試劑盒應(yīng)用于臨床。目的:以 GPC3 非抗體結(jié)合蛋白(non-antibody binding proteins,nABPs)的一員Affimer 和 GPC3 單克隆抗體(monoclonal antibody,mcAb,以簡(jiǎn)稱 Ab)為免疫原料,建立化學(xué)發(fā)光免疫分析法,評(píng)價(jià)其分析性能及其在臨床診斷中的價(jià)值,探討Affimer作為免疫分析試劑原料的可行性。方法:1.對(duì)團(tuán)隊(duì)篩選制備的Affimers和Abs進(jìn)行純化及鑒定,通過棋盤滴定法篩選獲取信噪比最佳的配對(duì)包被及標(biāo)記物質(zhì)。2.以Affimer-Ab雙夾心為反應(yīng)模式建立GPC3化學(xué)發(fā)光免疫分析法,優(yōu)化磁微球包被濃度、生物素與抗體的連接比例和吖啶酯標(biāo)記鏈霉親和素比例等反應(yīng)參數(shù)后,小試生產(chǎn)Affimer-Ab雜合GPC3化學(xué)發(fā)光免疫分析試劑。3.對(duì)小試試劑進(jìn)行最低檢測(cè)限、特異性、回收率等分析性能的評(píng)估,同期與GPC3雙抗體檢測(cè)試劑盒進(jìn)行分析性能的比對(duì)。4.用小試試劑盒及3個(gè)雙抗體檢測(cè)試劑盒檢測(cè)臨床血樣(HCC患者、肝硬化患者、肝內(nèi)膽管癌患者、慢性乙肝患者、慢性丙肝患者、健康體檢者患者、胃腸癌患者和肺癌患者)中GPC3水平。免疫組織化學(xué)法(IHC)檢測(cè)HCC組織標(biāo)本。曲線下面積和相關(guān)性分析等用于分析檢驗(yàn)試劑盒的檢測(cè)效能。結(jié)果:1.純化后所有原料均符合實(shí)驗(yàn)要求,確定Affimer-GPC3-22和Ab-7D11分別為本方法的包被和標(biāo)記物質(zhì)。2.確定了本方法最佳反應(yīng)條件:2步法的儀器運(yùn)行模式;Affimer-GPC3-22包被磁微球質(zhì)量比為1:80,生物素與抗體連接的質(zhì)量比為1:10,吖啶酯與鏈霉親和素標(biāo)記的質(zhì)量比為1:50;磁微球工作稀釋緩沖液pH為7.8;參考標(biāo)準(zhǔn)品加樣體織為60μL;包被磁微球、生物素化抗體和吖啶酯標(biāo)記鏈霉親和素最佳工作稀釋度分別為800μg/mL、1:250和1:500;繪制標(biāo)準(zhǔn)工作曲線,得到回歸方程為 y=3.95+1.03x(R=0.9999)。3.分析性能評(píng)價(jià)結(jié)果:本試劑最低檢測(cè)限為0.03ng/mL;特異性交叉反應(yīng)率為 0-0.002%;在線性范圍 0.03-600ng/mL 時(shí),R=0.9999;回收率在 91.80%-104.53%之間;批內(nèi)、批間精密度CV在6.06-8.98%之間;檢測(cè)樣本時(shí)不受膽紅素、溶血、乳糜微粒的干擾;效期穩(wěn)定性不少于12個(gè)月,開瓶穩(wěn)定性為14天。與商業(yè)化GPC3試劑盒比對(duì),本方法靈敏度適中、特異性好、線性范圍寬。4.本試劑正常參考值范圍為0-1.1ng/mL;HCC組GPC3血清水平明顯高于其他組(＞16倍,P值均小于0.001);術(shù)前GPC3表達(dá)水平明顯高于術(shù)后第一天和第七天,且呈逐步下降的趨勢(shì);四個(gè)試劑盒相關(guān)性均較差(r在-0.286至0.478之間);與IHC檢測(cè)結(jié)果比對(duì),本方法顯示良好的特異性和一致性(Kappa=0.655)。結(jié)論:上述結(jié)果表明本研究成功研制了 Affimer-Ab雜合GPC3化學(xué)發(fā)光免疫分析法,分析性能與臨床檢測(cè)結(jié)果顯示建立的方法在檢測(cè)血清GPC3上有著良好可靠性和準(zhǔn)確性,初步說明非抗體結(jié)合蛋白Affimer有望作為抗體的有效補(bǔ)充替代物用于免疫檢測(cè)試劑的研制。
[Abstract]:Phosphatidylinositol proteoglycan-3 (glypican-3,GPC3) is a valuable marker for the diagnosis of hepatocellular carcinoma (HCC). However, up to now, there is no reliable serological detection kit for clinical application. Aim: to establish chemiluminescence immunoassay with Affimer and GPC3 monoclonal antibodies (monoclonal antibody,mcAb,) of GPC3 non-antibody binding protein (non-antibody binding proteins,nABPs) as immune raw materials, to evaluate its analytical performance and its value in clinical diagnosis, and to explore the feasibility of Affimer as raw material for immunoassay. Method: 1. The Affimers and Abs prepared by team screening were purified and identified. The best pairing coating and labeling substance with signal-to-noise ratio (SNR) were obtained by chessboard titration. 2. Using Affimer-Ab double sandwich as reaction mode, GPC3 chemiluminescence immunoassay was established. After optimizing the concentration of magnetic microspheres, the ratio of biotin to antibody and the ratio of acridyl ester labeled streptavidin, Affimer-Ab hybrid GPC3 chemiluminescence immunoassay reagent was produced. 3. The minimum detection limit, specificity, recovery and other analytical performance of the small test reagent were evaluated, and the analytical performance of the small test reagent was compared with that of the GPC3 double antibody detection kit at the same time. 4. The levels of GPC3 in clinical blood samples (HCC patients, cirrhotic patients, intrahepatic bile duct cancer patients, chronic hepatitis B patients, chronic hepatitis C patients, healthy physical examination patients, gastrointestinal cancer patients and lung cancer patients) were measured by small test kit and three double antibody detection kits. HCC tissue samples were detected by (IHC). The area under the curve and correlation analysis are used to analyze and test the detection efficiency of the kit. Result: 1. After purification, all the raw materials met the experimental requirements, and Affimer-GPC3-22 and Ab-7D11 were determined to be the coating and labeling substances of this method, respectively. The optimum reaction conditions were determined as follows: the instrument operation mode of 2-step method, the mass ratio of Affimer-GPC3-22 coated magnetic microspheres was 1 鈮，
本文編號(hào)：2502825
[Abstract]:Phosphatidylinositol proteoglycan-3 (glypican-3,GPC3) is a valuable marker for the diagnosis of hepatocellular carcinoma (HCC). However, up to now, there is no reliable serological detection kit for clinical application. Aim: to establish chemiluminescence immunoassay with Affimer and GPC3 monoclonal antibodies (monoclonal antibody,mcAb,) of GPC3 non-antibody binding protein (non-antibody binding proteins,nABPs) as immune raw materials, to evaluate its analytical performance and its value in clinical diagnosis, and to explore the feasibility of Affimer as raw material for immunoassay. Method: 1. The Affimers and Abs prepared by team screening were purified and identified. The best pairing coating and labeling substance with signal-to-noise ratio (SNR) were obtained by chessboard titration. 2. Using Affimer-Ab double sandwich as reaction mode, GPC3 chemiluminescence immunoassay was established. After optimizing the concentration of magnetic microspheres, the ratio of biotin to antibody and the ratio of acridyl ester labeled streptavidin, Affimer-Ab hybrid GPC3 chemiluminescence immunoassay reagent was produced. 3. The minimum detection limit, specificity, recovery and other analytical performance of the small test reagent were evaluated, and the analytical performance of the small test reagent was compared with that of the GPC3 double antibody detection kit at the same time. 4. The levels of GPC3 in clinical blood samples (HCC patients, cirrhotic patients, intrahepatic bile duct cancer patients, chronic hepatitis B patients, chronic hepatitis C patients, healthy physical examination patients, gastrointestinal cancer patients and lung cancer patients) were measured by small test kit and three double antibody detection kits. HCC tissue samples were detected by (IHC). The area under the curve and correlation analysis are used to analyze and test the detection efficiency of the kit. Result: 1. After purification, all the raw materials met the experimental requirements, and Affimer-GPC3-22 and Ab-7D11 were determined to be the coating and labeling substances of this method, respectively. The optimum reaction conditions were determined as follows: the instrument operation mode of 2-step method, the mass ratio of Affimer-GPC3-22 coated magnetic microspheres was 1 鈮，
本文編號(hào)：2502825
本文鏈接：http://sikaile.net/yixuelunwen/jichuyixue/2502825.html
最近更新
教材專著
