Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method.
Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method.
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使用纳米金免疫学扩增和银色染色方法基于蛋白质CHIP测定法和基于蛋白质CHIP测定的人类乙型肝炎病毒和丙型肝炎病毒抗体的快速和同时检测。
DOI：
10.1186/1471-2334-5-53
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发表时间：
2005-07-06
影响因子：
3.7
通讯作者：
Zhai, JX
中科院分区：
文献类型：
--
作者：
Duan, LL;Wang, YF;Li, SSC;Wan, ZX;Zhai, JX
由乙型肝炎病毒和丙型肝炎病毒引起的病毒性肝炎是全世界主要的公共卫生问题。传统的检测方法包括基于聚合酶链反应（PCR）的检测和酶联免疫吸附试验（ELISA），这些方法既昂贵又耗时。在我们的实验中，采用纳米金免疫扩增和银染色（NIASS）方法的蛋白质芯片法快速同时检测HBV和HCV抗体。采用化学修饰的载玻片作为固体载体（称为芯片），在载玻片上分别固定HBsAg、HBeAg、HBcAg和HCVAg （NS3、NS5和核心抗原的混合物）等抗原。以胶体纳米金标记的葡萄球菌蛋白A （staphylococcal protein A， SPA）为指示剂，采用免疫金银染色增强技术对检测信号进行放大，在阵列斑点上产生肉眼可见的黑色图像。为了确定蛋白芯片法的检出限，我们构建了一组人IgG标记的模型阵列，并将模型阵列与不同浓度的抗IgG孵育。将305份先前用商用ELISA鉴定的血清样本分为4组，并在本试验中进行检测。我们制备了平均直径为15±2 nm的单分散球形纳米金颗粒。透射电镜观察到的纳米spa金胶体颗粒分布均匀、稳定。最佳银增强时间为8 ~ 12分钟。在我们的实验中，蛋白芯片可以检测血清中针对HBsAg、HBeAg、HBcAg和HCVAg的抗体，并且没有交叉反应。在模型阵列中，可以检测到低至3 ng/ml的抗igg。蛋白质芯片法与酶联免疫吸附测定法的结果无显著差异（P < 0.05）。结果表明，该方法可与血清学同时快速检测HBV和HCV抗体。
Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively. Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.
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影响因子：
3.9
作者：
Fan, CY;Jiang, L
通讯作者：
Jiang, L
影响因子：
--
作者：
QUINN, M;MILLS, G
通讯作者：
MILLS, G
影响因子：
5.1
作者：
Shad, J A;McHutchison, J G
通讯作者：
McHutchison, J G
影响因子：
12.7
作者：
Wang, YF;Pang, DW;Shen, JT
通讯作者：
Shen, JT
影响因子：
2.2
作者：
Huang, RP
通讯作者：
Huang, RP