Mutua Maurine Mumo, Kanoi Bernard N, Nyanjom Steven Ger, Musundi Sebastian, Makau Mark, Inoue Shingo, Khamadi Samoel Ashimosi, Gitaka Jesse, Wandera Ernest Apondi
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya.
Centre for Research in Infectious Diseases, Directorate of Research and Innovation, Mount Kenya University, P.O. Box 342-01000, Thika, Kenya.
Trop Med Health. 2025 May 13;53(1):69. doi: 10.1186/s41182-025-00756-y.
The coronavirus disease 2019 (COVID-19) pandemic underscored the global need for reliable diagnostic tools with quick turnaround time for effective patient management and mitigation of virus spread. This study aimed to express severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein and produce monoclonal antibodies (mAbs) against the expressed protein.
Following successful expression and purification of His-tagged SARS-CoV-2 N protein using a wheat germ cell-free protein expression system (WGCFS), BALB/c mice were immunized, and generated hybridomas screened for mAb production. Indirect and sandwich ELISA were used to screen the reactivity of the monoclonal antibody against both our recombinant antigen and commercial antigen. The mAbs were also assessed for their performance using RT-PCR confirmed positive samples with varying cycle threshold (CT) values and their specificity screened using virus isolates of other respiratory viruses.
Our mAb demonstrated high reactivity against our recombinant antigen, commercial antigen, SARS-CoV-2 Beta and Omicron variants. There was no significant difference in the binding affinity of our mAb and commercial mAb against the study recombinant (p = 0.12) and commercial (p = 0.072) antigens. Our mAb detected SARS-CoV-2 from clinical samples with varying CT values and exhibited no cross-reactivity against other respiratory viruses.
We successfully expressed SARS-CoV-2 N protein leveraging WGCFS in a resource-limited setting. Our mAb had a high binding affinity to the recombinant antigen, making it a suitable candidate for antigen detection kit development. Beyond diagnostics, the mAb holds potential for therapeutic applications as well as use in clinical and environmental surveillance platforms.
2019年冠状病毒病（COVID-19）大流行凸显了全球对可靠诊断工具的需求，这些工具需要快速周转时间，以实现有效的患者管理和减轻病毒传播。本研究旨在表达严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核衣壳蛋白，并产生针对该表达蛋白的单克隆抗体（mAb）。
使用小麦胚无细胞蛋白质表达系统（WGCFS）成功表达并纯化His标签的SARS-CoV-2 N蛋白后，对BALB/c小鼠进行免疫，并筛选产生单克隆抗体的杂交瘤。采用间接和夹心ELISA法筛选单克隆抗体对重组抗原和商业抗原的反应性。还使用RT-PCR确认的具有不同循环阈值（CT）值的阳性样本评估单克隆抗体的性能，并使用其他呼吸道病毒的病毒分离株筛选其特异性。
我们的单克隆抗体对重组抗原、商业抗原、SARS-CoV-2 Beta和Omicron变体表现出高反应性。我们的单克隆抗体和商业单克隆抗体对研究重组抗原（p = 0.12）和商业抗原（p = 0.072）的结合亲和力没有显著差异。我们的单克隆抗体从具有不同CT值的临床样本中检测到SARS-CoV-2，并且对其他呼吸道病毒没有交叉反应。
我们在资源有限的环境中利用WGCFS成功表达了SARS-CoV-2 N蛋白。我们的单克隆抗体对重组抗原有高结合亲和力，使其成为抗原检测试剂盒开发的合适候选者。除了诊断之外，该单克隆抗体在治疗应用以及临床和环境监测平台中也具有潜力。
