Affimer-抗体杂合化学发光免疫分析法的建立及其在血清磷脂酰肌醇蛋白聚糖-3检测中的应用研究
发布时间：2019-06-20 02:03
【摘要】：磷脂酰肌醇蛋白聚糖-3(glypican-3,GPC3)是一个有价值的肝细胞癌(HCC)诊断标志物,然而到目前为止,尚未有可靠的血清学检测试剂盒应用于临床。目的:以 GPC3 非抗体结合蛋白(non-antibody binding proteins,nABPs)的一员Affimer 和 GPC3 单克隆抗体(monoclonal antibody,mcAb,以简称 Ab)为免疫原料,建立化学发光免疫分析法,评价其分析性能及其在临床诊断中的价值,探讨Affimer作为免疫分析试剂原料的可行性。方法:1.对团队筛选制备的Affimers和Abs进行纯化及鉴定,通过棋盘滴定法筛选获取信噪比最佳的配对包被及标记物质。2.以Affimer-Ab双夹心为反应模式建立GPC3化学发光免疫分析法,优化磁微球包被浓度、生物素与抗体的连接比例和吖啶酯标记链霉亲和素比例等反应参数后,小试生产Affimer-Ab杂合GPC3化学发光免疫分析试剂。3.对小试试剂进行最低检测限、特异性、回收率等分析性能的评估,同期与GPC3双抗体检测试剂盒进行分析性能的比对。4.用小试试剂盒及3个双抗体检测试剂盒检测临床血样(HCC患者、肝硬化患者、肝内胆管癌患者、慢性乙肝患者、慢性丙肝患者、健康体检者患者、胃肠癌患者和肺癌患者)中GPC3水平。免疫组织化学法(IHC)检测HCC组织标本。曲线下面积和相关性分析等用于分析检验试剂盒的检测效能。结果:1.纯化后所有原料均符合实验要求,确定Affimer-GPC3-22和Ab-7D11分别为本方法的包被和标记物质。2.确定了本方法最佳反应条件:2步法的仪器运行模式;Affimer-GPC3-22包被磁微球质量比为1:80,生物素与抗体连接的质量比为1:10,吖啶酯与链霉亲和素标记的质量比为1:50;磁微球工作稀释缓冲液pH为7.8;参考标准品加样体织为60μL;包被磁微球、生物素化抗体和吖啶酯标记链霉亲和素最佳工作稀释度分别为800μg/mL、1:250和1:500;绘制标准工作曲线,得到回归方程为 y=3.95+1.03x(R=0.9999)。3.分析性能评价结果:本试剂最低检测限为0.03ng/mL;特异性交叉反应率为 0-0.002%;在线性范围 0.03-600ng/mL 时,R=0.9999;回收率在 91.80%-104.53%之间;批内、批间精密度CV在6.06-8.98%之间;检测样本时不受胆红素、溶血、乳糜微粒的干扰;效期稳定性不少于12个月,开瓶稳定性为14天。与商业化GPC3试剂盒比对,本方法灵敏度适中、特异性好、线性范围宽。4.本试剂正常参考值范围为0-1.1ng/mL;HCC组GPC3血清水平明显高于其他组(＞16倍,P值均小于0.001);术前GPC3表达水平明显高于术后第一天和第七天,且呈逐步下降的趋势;四个试剂盒相关性均较差(r在-0.286至0.478之间);与IHC检测结果比对,本方法显示良好的特异性和一致性(Kappa=0.655)。结论:上述结果表明本研究成功研制了 Affimer-Ab杂合GPC3化学发光免疫分析法,分析性能与临床检测结果显示建立的方法在检测血清GPC3上有着良好可靠性和准确性,初步说明非抗体结合蛋白Affimer有望作为抗体的有效补充替代物用于免疫检测试剂的研制。
[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，
本文编号：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，
本文编号：2502825
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