Evaluation of a safe and sensitive Spike protein-based immuno fluorescence assay for the detection of antibody responses to SARS-CoV
Evaluation of a safe and sensitive Spike protein-based immuno fluorescence assay for the detection of antibody responses to SARS-CoV
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DOI：
10.1016/j.jim.2004.10.012
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发表时间：
2005-01-01
影响因子：
2.2
通讯作者：
Kwang, J
中科院分区：
文献类型：
--
作者：
Manopo, I;Lu, LQ;Kwang, J
在此之前，我们已经鉴定出一段截短的抗原片段，命名为蛋白C[441到700个氨基酸(aa.)]。作为严重急性呼吸综合征冠状病毒(SARS-CoV)S蛋白(S)的免疫优势片段。我们现在已经成功地利用杆状病毒系统在果树链霉菌(SF-9)细胞中表达了蛋白C。用5份SARS恢复期血清和5份正常人血清对表达蛋白C的重组杆状病毒进行了鉴定。结果表明，蛋白C是抗SARS冠状病毒抗体的可靠抗原。我们的基于重组杆状病毒-SF-9系统的基于Spike蛋白的免疫荧光分析(IFA)进一步用新加坡SARS疫情期间收集的163份临床样本进行了评估，其中包括21份临床确诊的SARS患者血清、42份非SARS患者血清和100份正常血清。结果与商业SARS IFA试剂盒(德国EUROIMMUN)和在新加坡总医院进行的常规IFA测试进行了比较。21份SARS阳性血清标本均能被我们的IFA识别，其特异性和敏感性为100%，与两种基于病毒的IFA检测方法都是一致的。与传染性支气管炎病毒(IBV)和传染性胃肠炎病毒(TGEV)血清无交叉反应。因此，我们的基于Spike蛋白的IFA可以提供一个更安全的程序，可以在BSL-2实验室进行，因为它可以模拟整个基于病毒的IFA，而不会损失任何敏感性和特异性。它也比整个基于病毒的IFA更具用户友好性和成本效益。(C)2004爱思唯尔B.V.保留所有权利。
previously, we have identified a truncated antigenic fragment named protein C [441 to 700 amino acids (a.a.)] as the immunodominant fragment of Spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We have now successfully expressed protein C using the baculovirus system in S. frugiperda (Sf-9) cells. This recombinant baculovirus expressing protein C was first characterized using five SARS convalescent human sera and five normal human sera. The results showed that protein C is ail authentic antigen against SARS-CoV antibody. Our Spike protein-based immunoflourescence assay (IFA) based on this recombinant baculovirus-Sf-9 system was further assessed with a panel of 163 clinical samples collected during the SARS epidemic in Singapore, which include samples from 21 clinically confirmed SARS, 42 non-SARS patient sera, and 100 normal sera. The results were compared to a commercial SARS IFA kit (EUROIMMUN, Germany) and a conventional IFA test performed in Singapore General Hospital. All of the 21 SARS-positive serum samples Could be recognized by our IFA, giving a specificity and sensitivity of 100%, which was compatible with both whole virus-based IFA assays. No cross-reactivity with serum samples against infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV) were detected ill our assays. Thus, our Spike protein-based IFA Could offer a safer procedure which can be performed in a BSL-2 laboratory as it could mimic the whole virus based-IFA without any loss of sensitivity and specificity. It is also more User-friendly and cost-effective than the whole virus-based IFA. (C) 2004 Elsevier B.V. All rights reserved.