Elgh F, Lundkvist A, Alexeyev O A, Wadell G, Juto P
Department of Virology, University of Umeå, Sweden.
J Virol Methods. 1996 May;59(1-2):161-72. doi: 10.1016/0166-0934(96)02042-3.
Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU). The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man. We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE. Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed. The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies. The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94. A recombinant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE. ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity as compared to the full length recombinant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes. Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions.
流行性肾病（NE）是欧洲肾综合征出血热的主要形式，由汉坦病毒普马拉血清型（PUU）引起。已证明PUU病毒核衣壳蛋白（N）在实验动物和人类中均具有高度免疫原性。我们旨在确定在体液免疫反应中重要的结构域，并利用这些信息开发一种用于NE血清学诊断的特异性和敏感性酶联免疫吸附测定（ELISA）。构建了包含编码PUU病毒N（PUU rN）的重叠基因片段的大肠杆菌多组氨酸融合蛋白表达载体。用多克隆抗体和单克隆抗体通过免疫印迹和ELISA检测所得基因产物。PUU rN的主要抗原区域位于氨基酸（aa）7至94之间。含有PUU病毒N的aa 7-137（PUU rN delta 5）的重组融合蛋白用于检测NE中的特异性IgG和IgM反应。基于PUU rN delta 5的ELISA被发现与通过ELISA检测全长重组PUU rN相比，在NE的急性血清学诊断和血清流行病学筛查目的方面具有相同的敏感性和特异性。此外，由于该蛋白在水溶液中的溶解度更高，因此比全长PUU rN更易于处理。
