Lam Athene Hoi-Ying, Cai Jian-Piao, Leung Ka-Yi, Zhang Ricky-Ruiqi, Liu Danlei, Fan Yujing, Tam Anthony Raymond, Cheng Vincent Chi-Chung, To Kelvin Kai-Wang, Yuen Kwok-Yung, Hung Ivan Fan-Ngai, Chan Kwok-Hung
Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China.
Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China.
Diagnostics (Basel). 2021 Dec 13;11(12):2346. doi: 10.3390/diagnostics11122346.
Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.
免疫荧光法是一种用于呼吸道病毒的传统诊断方法，可实现快速、简单且准确的诊断，具有直接可视化目标抗原和进行质量评估的特定优势。本研究旨在评估间接免疫荧光法作为一种针对鼻咽拭子（NPS）中SARS-CoV-2抗原的内部诊断方法的潜力。使用了三种从小鼠体内产生的一抗进行免疫荧光染色，包括针对SARS-CoV核衣壳蛋白的单克隆抗体，以及针对SARS-CoV-2核衣壳蛋白和SARS-CoV-2刺突蛋白受体结合域的多克隆抗体。用这三种抗体对29例COVID-19患者和20名未感染个体的NPS细胞涂片以及病毒培养细胞进行染色。使用免疫荧光显微镜鉴定具有阳性信号的呼吸道上皮细胞。在测试的三种抗体中，针对SARS-CoV-2 N蛋白的多克隆抗体具有最高的敏感性和特异性，在RT-PCR确诊的29例COVID-19病例中检测出17例，并且除SARS-CoV外，与其他测试病毒无交叉反应。尽管准确性受样本质量和呼吸道上皮细胞数量的限制，但检测针对SARS-CoV-2 N蛋白的病毒感染细胞可识别受感染个体。证明了免疫荧光法作为一种简单诊断方法的潜力，通过将针对SARS-CoV-2的抗体纳入临床使用的多重免疫荧光检测板（如用于呼吸道病毒检测）中，可以在COVID-19流行率较低且疫情结束后仍对SARS-CoV-2进行额外的常规诊断和监测检测。
