Carattoli Alessandra, Di Bonito Paola, Grasso Felicia, Giorgi Colomba, Blasi Francesco, Niedrig Matthias, Cassone Antonio
Department of Infectious, Parasitic and Immuno-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
J Med Virol. 2005 Jun;76(2):137-42. doi: 10.1002/jmv.20338.
A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.
一种新型冠状病毒（SARS-CoV）是严重急性呼吸综合征（SARS）的病原体。鉴于血清学检测在SARS诊断中发挥的关键作用，开发了一种基于SARS-CoV重组抗原的内部ELISA。SARS-CoV核衣壳蛋白（N）、三个N片段（N1、N2和N3）以及膜蛋白（M2）的病毒内区域被克隆并在大肠杆菌中表达为组氨酸标签蛋白。使用来自SARS患者的六份参考血清，以每种重组蛋白作为包被抗原，通过ELISA检测病毒特异性IgG。在所有SARS阳性血清中均检测到高滴度阳性反应。该检测方法的特异性似乎很高，因为在20名健康受试者和73名患有非SARS下呼吸道感染的患者血清中未检测到阳性反应。还在小鼠和兔子中产生了针对SARS-CoV以及重组蛋白N、N1、N2、N3和M2的特异性超免疫血清。这些血清的特异性通过对SARS-CoV感染和未感染细胞的生物芯片进行免疫细胞化学检测得以证实。
