Yuan Fangfeng, Chen Chi, Covaleda Lina M, Martins Mathias, Reinhart Jennifer M, Sullivan Drew R, Diel Diego G, Fang Ying
bioRxiv. 2023 Mar 13:2023.03.11.532204. doi: 10.1101/2023.03.11.532204.
The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals.
Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的全球大流行对公共卫生构成重大威胁。除人类外，SARS-CoV-2还可感染多种动物物种。迫切需要高灵敏度和特异性的诊断试剂及检测方法，以便快速检测并实施动物感染防控策略。在本研究中，我们首先开发了一组针对SARS-CoV-2核衣壳（N）蛋白的单克隆抗体（mAb）。为检测多种动物物种中的SARS-CoV-2抗体，我们开发了一种基于单克隆抗体的阻断ELISA（bELISA）。使用一组已知感染状态的动物血清样本进行检测验证，得到的最佳抑制率（PI）截断值为17.6%，诊断灵敏度为97.8%，诊断特异性为98.9%。该检测方法具有很高的重复性，其批间、批内和板内变异系数分别较低（7.23%、6.95%和5.15%）。对实验感染猫随时间收集的样本进行检测表明，bELISA能够在感染后7天最早检测到血清转化。随后，bELISA被应用于检测有类似COVID-19症状的宠物，在两只狗中检测到了特异性抗体反应。本研究中产生的单克隆抗体组为SARS-CoV-2诊断和研究提供了有价值的工具。基于单克隆抗体的bELISA为动物COVID-19监测提供了一种血清学检测方法。
抗体检测通常用作检测感染后宿主免疫反应的诊断工具。血清学（抗体）检测通过提供病毒暴露史来补充核酸检测，无论感染是否出现症状或为无症状感染。对COVID-19的血清学检测需求很高，尤其是在有疫苗可用时。它们对于确定人群中病毒感染的流行率以及识别已感染或接种疫苗的个体很重要。ELISA是一种简单且实际可靠的血清学检测方法，可在监测研究中进行高通量检测。有几种COVID-19 ELISA试剂盒可供使用。然而，它们大多是为人样本设计的，间接ELISA形式需要物种特异性二抗。本文描述了一种适用于所有物种的基于单克隆抗体的阻断ELISA的开发，以促进动物中COVID-19的检测和监测。
