新型免疫电极法测定乙型肝炎表面抗原

本文报道一种新型的乙型肝炎抗体膜片。测试时,仅需将25μl被检血清滴注于该膜上,经孵育与洗涤即可制得抗原与抗体复合物膜。用复合物膜组装成免疫电极,测定血清样品中乙型肝炎表面抗原含量。电极的线性范围是20～320ng/ml,方法的日间相对标准偏差为10.78％(n=4,m=5),对血清中可能存在的一些其它抗原具有较好的选择性。105例临床血清样本,本法与酶联免疫吸附分析法测定结果符合率为86％。乙肝抗体膜片系一次性使用,4℃可保存半年以上。     

2-氨基-3-羟基吡啶-过氧化氢-辣根过氧化物酶伏安酶联免疫体系分析人血清癌胚抗原

以氮杂环化合物为电化学分析底物的2-氨基-3-羟基吡啶-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫体系测定人血清癌胚抗原(CEA).HRP催化H2O2氧化2-氨基-3-羟基吡啶的酶促反应产物,在缓冲液中-0.36 V处产生一个灵敏的伏安还原峰,借助此峰可以测定游离的HRP,进而可用于以HRP为标记物的酶联免疫分析.对酶促反应条件和测定条件的优化反应条件为:以B-R缓冲液(pH 6.0)为反应介质,在10 mL总反应液中含有1.0 mL 0.2 mol/L B-R缓冲液、3.0 mL 8.0 mmol/L 2-氨基-3-羟基吡啶溶液以及1.5 mL 0.5 mmol/L H2O2溶液,反应温度37 ℃,反应时间30 min.最佳测定条件为:B-R缓冲液(pH 7.0)为支持电解质,在10 mL总测定溶液中含有5 mL上述总反应液、1.0 mL 0.2 mol/L B-R缓冲液.测定仪器条件:起始电位0.00 V,终止电位-0.80 V,电位扫描速度400 mV/s,滴汞静止时间7 s.在最佳的反应条件和测定条件下,新体系测定游离HRP的线性范围为4.0×10-4～1.0 μg/L; 对HRP的检出限为0.12 ng/L.新体系对CEA测定的线性范围为0.50～80.0 μg/L; 检出限为0.50 μg/L.为经典ELISA法的检出限的1/10.     

Enhanced surface plasmon resonance by Au nanoparticles immobilized on a dielectric SiO2 layer on a gold surface

This paper introduces strategies for enhancement of a surface plasmon resonance (SPR) signal by adopting colloidal gold nanoparticles (AuNPs) and a SiO₂ layer on a gold surface. AuNPs on SiO₂ on a gold surface were compared with an unmodified gold surface and a SiO₂ layer on a gold surface with no AuNPs attached. The modified surfaces showed significant changes in SPR signal when biomolecules were attached to the surface as compared with an unmodified gold surface. The detection limit of AuNPs immobilized on a SPR chip was 0.1 ng mL⁻¹ for the prostate-specific antigen (PSA), a cancer marker, as measured with a spectrophotometer. Considering that the conventional ELISA method can detect ∼10 ng mL⁻¹ of PSA, the strategy described here is much more sensitive (∼100 fold). The enhanced shift of the absorption curve resulted from the coupling of the surface and particle plasmons by the SiO₂ layer and the AuNPs on the gold surface.     

化学发光免疫分析检测人血清中的癌胚抗原

基于辣根过氧化物酶(HRP)催化H2O2氧化3-(4-羟苯基)丙酸(PHPPA),生成能发荧光的3-(4-羟苯基)丙酸二聚体.在乙腈介质中,在增强剂眯唑参与下,与双[2,4,6-三氯苯基]草酸酯(TCPO)和H2O2反应产生强化学发光.用辣根过氧化物酶(HRP)标记癌胚抗原(CEA)单克隆抗体,通过CEA的双抗夹心免疫...     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

测试生物分子的新方法—伏安免疫法

本文综述了迄今为止国外和本实验室在伏安免疫法新领域的研究工作。阐述了伏安免疫法的发展和基本原理,讨论了伏安免疫分析的各种可行方法和前景。     

纳米孔技术对PD-1抗原抗体结合检测的实验研究

肿瘤的免疫疗法是利用人体自身的免疫系统去治疗肿瘤的一种方法,程序性死亡受体1（PD-1）是肿瘤免疫疗法中的一种免疫检查点,利用PD-1或PD-L1的单克隆抗体,可以阻断PD-1/PD-L1信号通路,恢复T细胞的免疫杀伤功能,实现肿瘤的免疫治疗.为了研究PD-1抗体药物与人体PD-1抗原的结合有效性,以PD-1蛋白质分子为实验对象,用纳米孔传感器件开展了PD-1、PD-1抗体及其结合物的辨识.分别对纯PD-1蛋白质分子和其抗体分子进行了纳米孔过孔实验研究,发现PD-1及其抗体的相对堵塞电流幅值比分别为0.00404和0.01297,实现了抗原抗体分子的区分.并对Si₃N₄纳米孔内壁进行了PD-1抗体修饰,再将PD-1蛋白质分子通过经抗体修饰后的纳米孔,以期实现结合物的检测,实验结果显示部分抗原抗体实现了特异性结合,剩下部分呈游离状,所以纳米孔技术可实现抗原抗体结合物的区分.未来,纳米孔有望成为无标志检测药物有效性的一种新手段.