Kyosei Yuta, Namba Mayuri, Makioka Daiki, Kokubun Ayumi, Watabe Satoshi, Yoshimura Teruki, Sasaki Tadahiro, Shioda Tatsuo, Ito Etsuro
Department of Biology, Waseda University, Tokyo 162-8480, Japan.
Waseda Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
Microorganisms. 2021 Oct 25;9(11):2214. doi: 10.3390/microorganisms9112214.
To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10 moles/assay for recombinant S1 proteins and 2.6 × 10 RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 10 RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19.
为帮助控制2019年冠状病毒病（COVID-19）的全球大流行，我们开发了一种针对引起该感染的病毒——严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的诊断方法。我们通过结合夹心酶联免疫吸附测定（ELISA）和硫代烟酰胺腺嘌呤二核苷酸（thio-NAD）循环反应应用了一种超灵敏方法来定量刺突S1蛋白。对于重组S1蛋白，检测限（LOD）为2.62×10摩尔/测定，对于紫外线B灭活病毒，检测限为2.6×10 RNA拷贝/测定。我们已经表明，用于核衣壳蛋白的超灵敏ELISA可以在10 RNA拷贝/测定水平检测紫外线B灭活病毒，而SARS-CoV-2的核衣壳蛋白很难与传统冠状病毒和SARS-CoV中的核衣壳蛋白区分开来。因此，仅针对核衣壳蛋白的抗原检测不足以确定病毒特异性。因此，建议结合针对刺突蛋白和核衣壳蛋白的检测，以提高COVID-19抗原检测的检测灵敏度和测试准确性。综上所述，我们目前的研究通过结合用于核衣壳蛋白的超灵敏ELISA来进行S1检测，为COVID-19提供了一种超灵敏、抗原特异性的检测方法。
