聚苯乙烯免疫检测球珠的表层改性及其性能

<正> 随着免疫检测在生物医学研究及临床诊断中的发展和应用,七十年代后期由高聚物制成的乳胶或球珠作为免疫载体亦得到较快的发展;八十年代以来有多种产品问世,其中聚苯乙烯球珠及检测板、盒国外已有专厂生产。 由于聚苯乙烯球珠对免疫体、酶等的固定系物理吸附,在制备及检测过程中,这些蛋     

Potentiometric Immunosensor Based on Immobilization of Hepatitis B Surface Antibody on Platinum Electrode Modified Silver Colloids and Polyvinyl Butyral as Matrixes

A highly sensitive immunosensor based on immobilization of hepatitis B surface antibody (HBsAb) on platinum electrode (Pt) modified silver colloids and polyvinyl butyral (PVB) as matrixes has been developed for potentiometric immunoanalysis to detect hepatitis B surface antigen (HBsAg) in this study. HBsAb molecules were immobilized successfully on nanometer‐sized silver colloid particles associated with polyvinyl butyral on a platinum electrode surface. The modification procedure was electrochemically monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The HBsAb‐silver‐PVB‐modified electrode exhibited direct electrochemical behavior toward HBsAg. The factors influencing the performance of the resulting immunosensor were studied in detail. More than 94.7% of the results of human serum samples obtained by this method were in agreement with those obtained by enzyme‐linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited a sigmoid curve with log HBsAg concentration, high sensitivity (39.8 mV/decade), wide linear range from 16.0 to 800 ng mL^?1 with a detection limit of 3.6 ng mL^?1, fast potentiometric response (<3 min) and long‐term stability (>4 months). The response mechanism of the immunosensors was also studied with AC impedance techniques.     

乙肝患者头发微量元素分析

应用装有ＥＰＭ－８１０Ｑ电子探针扫描分析电镜，对５０例乙肝患者头发中Ｍｇ、Ａｌ、Ｃｄ、Ｃａ、Ｃｒ、Ｍｎ、Ｆｅ、Ｃｕ、Ｚｎ、Ｐｂ、Ｓｅ１１种元素含量进行分析，并与健康人头发进行比较。结果表明：乙肝组中Ｚｎ含量低于健康组，Ｃｕ、Ｃｄ、Ｆｅ含量高于健康组（Ｐ〈０．０１）。     

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self‐assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost‐effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE‐EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE‐EE method are monodisperse with polydispersity values of ～15% and ～13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE‐EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ～84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.     

利用无标记的分子信标及核酸染料SYBR Green Ⅰ检测乙肝病毒

本文以野生型的乙型肝炎病毒(HBV)核酸片段为研究对象,利用无标记的分子信标及核酸染料SYBR Green I,建立了一种高灵敏、高选择性的特定序列核酸检测方法.在优化条件下,目标DNA浓度为4×10-11~400×10-11 mol/L之间时,SYBR Green I的荧光强度(ΔI)与目标DNA的浓度(C)具有良好的线性关系,其拟合的回归方程为ΔI=1.9556 C+31.4659(R2=0.9956),方法检测限(3ζ)为2×10-11 mol/L.该方法操作简单、检测速度快、灵敏度高、重现性好、检出限低.利用该方法,结合不对称PCR技术,实现了对HBV的定量检测.     

分子信标用于不对称PCR检测乙型肝炎病毒

为了缓解竞争杂交对分子信标检测的影响，将分子信标与不对称聚合酶链式反应(PCR)联用进行血清中乙型肝炎病毒(HBV)的检测。并采用更为直接的荧光方法，将不对称PCR扩增中的引物浓度比确定为5：1，该结果与电泳方法得到的结果有所不同。文中结合对称及不对称PCR过程中荧光强度的变化情况，对其原因进行了详细的探讨。分子信标与不对称PCR联用，可以专一地检测出血清中HBV的存在，与分子信标／对称PCR相比．其检测效果明显增强。     

HCV全基因组培养细胞的比较蛋白组学研究

利用比较蛋白质组技术研究了转染丙型肝炎病毒(Hepatitis C virus, HCV)全基因组的人肝癌细胞系Huh7细胞模型中蛋白质表达谱的变化, 建立了Huh7-HCV的双向凝胶电泳蛋白质表达图谱和数据库. 通过双向凝胶电泳分离和图像分析, 对表达差异2倍以上蛋白质点进行了胶内酶解和MALDI-TOF MS鉴定. 得到包括与细胞骨架蛋白、细胞周期、凋亡和信号转导等相关的14个蛋白质, 并且用Western blot验证了热休克蛋白70的蛋白质组研究结果. 利用HCV全基因组培养系统, 采用蛋白质组学技术, 为研究HCV病毒和宿主细胞相互作用提供了新的实验数据, 为深入研究HCV病毒复制和分子致病机理奠定了基础.     

The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.