Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins
Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins
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DOI：
10.1128/jcm.39.1.1-7.2001
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发表时间：
2001-01-01
影响因子：
9.4
通讯作者：
Kurane, I
中科院分区：
文献类型：
--
作者：
Saijo, M;Niikura, M;Kurane, I
利用杆状病毒系统将埃博拉病毒（EBO）全长核蛋白（NP）表达为his标记的重组蛋白（His-EBO-NP）。EBO和马尔堡病毒（MBG） NPs的羧基末端半段在大肠杆菌系统中以谷胱甘肽转移酶标记的重组蛋白表达。采用Western blotting和酶联免疫吸附法（ELISA）测定EBO和MBG NPs上的抗原区域位于c端半。EBO和MBG NPs的c端110和102个氨基酸分别具有较强的抗原性。重组杆状病毒感染后，EBO的全长NP在昆虫细胞中有较强的表达，而MBG的全长NP表达较弱。我们利用His-EBO-NP和EBO和MBG的NPs的c端半段作为抗原，开发了免疫球蛋白G (IgG) ELISA。我们利用从EBO和MBG患者收集的人血清，评估了IgG ELISA检测EBO和MBG IgG抗体的能力。重组NPs的IgG ELISA检测EBO和MBG抗体具有较高的敏感性和特异性。结果表明，用EBO和MBG重组蛋白制备的ELISA系统是诊断EBO和MBG感染和进行血清流行病学现场研究的有价值的工具。
The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathioneS-transferase-tagged recombinant proteins in anEscherichia colisystem. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.