Toft Casey J, Bourquin Rebecca A, Sorenson Alanna E, Horwood Paul F, Druce Julian D, Schaeffer Patrick M
Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD 4811, Australia.
Veterinary Sciences, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD 4811, Australia.
Talanta Open. 2023 Aug;7:100187. doi: 10.1016/j.talo.2023.100187. Epub 2023 Jan 21.
Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID) assay. Of note, TCID discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.
积极的诊断检测仍然是医疗保健和老年护理机构预防SARS-CoV-2在脆弱人群中传播的不可或缺的策略。首选的诊断平台已转向新冠病毒快速抗原检测（RAT），以识别传染性最强的个体。因此，RAT的生产速度比以往任何时候都要快，但缺乏相关的定量分析，无法提供绝对分析灵敏度信息，这使得制造商难以保持高批次间的重现性，终端用户也难以准确比较不同品牌以供决策。在此，我们描述了一种新的参考标准，用于使用重组绿色荧光蛋白标记的核衣壳蛋白（NP-GFP）测量和比较RAT的分析灵敏度。重要的是，我们表明绿色荧光蛋白标签不会干扰NP检测，并具有几个优点，可实现高效的简化蛋白表达和纯化，以及用于蛋白溶解度和稳定性生产后评估的更快、更便宜、更灵敏的质量控制措施。使用NP-GFP作为参考标准对十种商用新冠病毒RAT进行了评估和排名。用NP-GFP测定的所选设备的分析灵敏度数据与制造商使用半数组织培养感染剂量（TCID）测定法报告的数据不相关。值得注意的是，此前已有关于TCID不一致的报道。综上所述，我们的结果凸显了迫切需要一种可靠的参考标准来评估和衡量RAT设备的分析灵敏度。NP-GFP作为一种参考标准很有前景，它将确保RAT的性能准确传达给医疗服务提供者和公众。
