Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361-87 Avenue, Edmonton, Alberta, Canada.
J Virol Methods. 2013 Jan;187(1):72-8. doi: 10.1016/j.jviromet.2012.09.006. Epub 2012 Sep 17.
The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037μg/ml (p<0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019μg/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS.
严重急性呼吸系统综合征冠状病毒（SARS-CoV）的刺突蛋白被认为是介导受体相互作用和免疫识别的主要目标，因此被认为是疫苗设计的主要目标。刺突蛋白在病毒进入、病毒受体相互作用和病毒嗜性中起着重要作用。对 SARS 的敏感诊断对于控制人类疾病至关重要。为了开发单克隆抗体和双特异性单克隆抗体，我们生产并纯化了重组 SARS-CoV S1 抗原。分别将分泌抗 S1 抗体的杂交瘤 F26G18 和 P136.8D12 与 YP4 杂交瘤融合，生成四聚体。夹心 ELISA 由 F26G18 作为包被抗体，生物素化 F26G18 作为检测抗体组成，检测限为 0.037μg/ml（p<0.02）。当使用 P136.8D12 作为包被抗体和生物素化 F26G18 作为检测抗体时，也发现了相同的检测限。然而，当使用双特异性单克隆抗体（F157）作为检测抗体时，灵敏度提高（检测限为 0.019μg/ml）。总之，本研究中描述的方法允许通过夹心 ELISA 用双特异性单克隆抗体敏感地检测重组 SARS 刺突蛋白，并可用于诊断疑似 SARS 的患者。
