Abstract
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 × 10−19 moles/assay for recombinant S1 proteins and 2.6 × 106 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 104 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.
Highlights
Coronavirus disease 2019 (COVID-19) has significantly changed people’s lifestyles, economics, and medical service systems throughout the world
The most important improvement is that the new antibodies used in the present study can recognize the ultraviolet B (UVB)-inactivated SARS-CoV-2, whereas the antibodies used in the previous recognize the UVB-inactivated SARS-CoV-2, whereas the antibodies used in the previous study [17] cannot recognize these viruses
It is difficult to detect the spike proteins of SARS-CoV-2 [13], but the simultaneous detection of nucleocapsid proteins and spike proteins is suitable for COVID-19 antigen tests [19]
Summary
Coronavirus disease 2019 (COVID-19) has significantly changed people’s lifestyles, economics, and medical service systems throughout the world. The widespread effects of the pandemic have provided opportunities for researchers to urgently develop useful diagnostics, effective vaccines, and specific drugs, for this pandemic, but future pandemics as well. We aimed to improve antigen tests as an important diagnostic modality [1]. The severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) causing COVID-19 contains a single-stranded RNA (ssRNA) genome and four major proteins (spike proteins, nucleocapsid proteins, envelope proteins, and membrane proteins) [2]. The entry of SARS-CoV-2 into host cells is mediated by a transmembrane structural glycoprotein (i.e., spike protein) containing the N-terminal S1 fragment and the. The spike protein is a clove-shaped structure that protrudes
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