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Summary: The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

The spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and receptor-binding activity of this protein considering the emergence of highly infectious viral variants. In this study, we attempted to express the receptor-binding region (RBD) of SARS-CoV-2 SP containing disulfide bonds using the wheat germ cell-free protein synthesis system. By adding protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase (ERO1α) to the translational reaction mixture, we succeeded in synthesizing a functionally intact RBD protein that can interact with ACE2. Using this RBD protein, we have developed a high-throughput AlphaScreen assay to evaluate the RBD–ACE2 interaction, which can be applied for drug screening and mutation analysis. Thus, our method sheds new light on the structural and functional properties of SARS-CoV-2 SP and has the potential to contribute to the development of new COVID-19 therapeutics.