Alexeyev O A, Elgh F, Ahlm C, Stigbrand T, Settergren B, Wadell G, Juto P
Department of Virology, University of Umea, Sweden.
Am J Trop Med Hyg. 1996 Apr;54(4):367-71. doi: 10.4269/ajtmh.1996.54.367.
An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.
开发了一种酶联免疫吸附测定（ELISA）法，用于检测细胞培养物中的不同汉坦病毒抗原，即普马拉（PUU）、汉滩（HTN）和多布拉瓦（DOB）病毒。该测定基于利用山羊抗IgM抗体将人血清免疫球蛋白M（IgM）抗体结合到固相上。捕获的IgM抗体存在于两名患者急性期血清中：一名在瑞典感染，另一名在波斯尼亚感染。通过广泛反应的多克隆兔抗PUU重组核衣壳蛋白抗血清检测与人抗PUU和抗DOB/HTN IgM抗体结合到固相上的抗原。当用作捕获抗体时，IgM同种型被证明比IgG至少高效五倍。通过用PUU重组核蛋白抗原滴定测量，PUU抗原ELISA的灵敏度约为0.5 ng/ml。通过PUU-ELISA在48小时后以及通过免疫荧光测定在96小时后在组织培养物中观察到细胞相关的PUU抗原。当测试区分PUU、DOB和HTN病毒的能力时，发现了显著差异：瑞典血清以高滴度检测到PUU抗原，而对DOB和HTN无反应性；波斯尼亚血清以高滴度检测到DOB和HTN，但对PUU反应性低。还测试了该方法在检测棕背鼠（黄毛鼠）肺中PUU抗原的实用性。在从流行性肾病患者周围捕获的59只动物中，三只在PUU-ELISA中呈高活性阳性，但在DOB/HTN-ELISA中反应性低。结论是已开发出一种灵敏的ELISA法，用于检测细胞培养物和棕背鼠肺中的不同汉坦病毒。
