Joint National Laboratory for Antibody Drug Engineering, Clinical Laboratory of the First Affiliated Hospital, School of Medicine, Henan University, Kaifeng, 475004, People's Republic of China.
Center of Smart Laboratory and Molecular Medicine, Jiangjin Hospital, School of Medicine, Chongqing University, Chongqing, 400044, People's Republic of China.
Int J Nanomedicine. 2023 Jun 12;18:3125-3139. doi: 10.2147/IJN.S408921. eCollection 2023.
INTRODUCTION: Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. METHODS: Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. RESULTS: RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 μg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. CONCLUSION: The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. CLINICAL TRIAL REGISTRATION: Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.
简介：中和抗体（NAb）对于预防再次感染 SARS-CoV-2 和 COVID-19 的复发至关重要；然而，由于在常规实验室环境中缺乏实用且有效的 NAb 检测方法，疫苗接种和感染后 NAb 的形成仍然是一个谜。在这项研究中，我们开发了一种方便的侧向流动分析方法，可在 20 分钟内快速准确地测量血清 NAb 水平。 方法：受体结合域片段结晶（RBD-Fc）和血管紧张素转换酶 2-组氨酸标签（ACE2-His）分别通过克隆 9 和人胚肾 293T 的真核表达系统表达。然后，合成胶体金并与 ACE2 偶联。在优化了各种操作参数后，构建了 NAb 侧向流动分析。随后，系统评估了其检测限、特异性和稳定性，并分析了临床样本以验证其临床可行性。 结果：RBD-Fc 和 ACE2-His 的纯度分别为 94.01%和 90.05%。合成的胶体金具有均匀的分布，平均粒径为 24.15±2.56nm。该检测方法的检测限为 2μg/mL，在 684 份未感染的临床样本中，其灵敏度为 97.80%，特异性为 100%。通过评估 356 份感染个体的样本，我们发现该检测方法与常规酶联免疫吸附试验的总符合率为 95.22%，并且我们注意到，16.57%（59/356）的个体在感染后（ELISA 和该检测方法均）仍未产生 NAb。通过该检测方法，所有上述检测都可以在 20 分钟内通过肉眼获得结果，无需任何额外的仪器或设备。 结论：该检测方法可快速可靠地检测感染后抗 SARS-CoV-2 NAb，结果为有效预防和控制 SARS-CoV-2 提供了有价值的数据。 临床试验注册：血清和血液样本的使用得到了河南大学生物医学伦理分委员会的批准，临床试验注册号为 HUSOM-2022-052。我们确认本研究符合《赫尔辛基宣言》。
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