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      An ultrasensitive ELISA to Assay Femtomolar Level SARS-CoV-2 Spike Antigen Based on Specific Peptide and Tyramine Signal Amplification

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          Abstract

          The SARS-CoV-2 spreading rapidly has aroused catastrophic public healthcare issues and economy crisis worldwide. It plays predominant role to rapidly and accurately diagnose the virus for effective prevention and treatment. As an abundant transmembrane protein, spike protein (SP) is one of the most valuable antigenic biomarkers for diagnosis of COVID-19. Herein a phage expression of WNLDLSQWLPPM peptide specific to SARS-CoV-2 SP was screened. Molecular docking revealed that the isolated peptide binds to major antigenic epitope locating at S2 subunit with hydrogen bonding. Taking the specific peptide as antigen sensing probe and tyramine signal amplification (TSA), an ultrasensitive "peptide-antigen-antibody" ELISA (p-ELISA) was explored, by which the limit of detection (LOD) was 14 fM and 2.8 fM SARS-CoV-2 SP antigen for first TSA and secondary TSA, respectively. Compared with the LOD by the p-ELISA by direct mode, the sensitivity with 2 nd TSA enhanced 100 times. Further, the proposed p-ELISA method can detect SARS-CoV-2 pseudoviruses down to 10 and 3 TCID 50/mL spiked in healthy nasal swab sample with 1 st TSA and 2 nd TSA, separately. Thus, the proposed p-ELISA method with TSA is expected to be a promising ultrasensitive tool for rapidly detecting SARS-CoV-2 antigen to help control the infectious disease.

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          Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

          Daniel Wrapp, Nianshuang Wang, Kizzmekia S. Corbett … (2020)
          Structure of the nCoV trimeric spike The World Health Organization has declared the outbreak of a novel coronavirus (2019-nCoV) to be a public health emergency of international concern. The virus binds to host cells through its trimeric spike glycoprotein, making this protein a key target for potential therapies and diagnostics. Wrapp et al. determined a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy. Using biophysical assays, the authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor. They also tested three antibodies known to bind to the SARS-CoV spike protein but did not detect binding to the 2019-nCoV spike protein. These studies provide valuable information to guide the development of medical counter-measures for 2019-nCoV. Science, this issue p. 1260
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            Structure, Function, and Evolution of Coronavirus Spike Proteins

            Fang Li (2016)
            The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells.
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              Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

              Katharine Crawford, Rachel Eguia, Adam Dingens … (2020)
              SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Sens Actuators B Chem
                Journal ID (iso-abbrev): Sens Actuators B Chem
                Title: Sensors and Actuators. B, Chemical
                Publisher: Elsevier B.V.
                ISSN (Print): 0925-4005
                ISSN (Electronic): 0925-4005
                Publication date PMC-release: 29 March 2023
                Publication date (Electronic): 29 March 2023
                Electronic Location Identifier: 133746
                Affiliations
                [0005]Institute for Chemical Biology & Biosensing, and College of Life Sciences, Qingdao University, 308 Ningxia Road, Qingdao 266071, China
                Author notes
                [* ]Corresponding author. E-mail:
                [1]

                These authors contributed equally to this work.

                Article
                Publisher Item ID: S0925-4005(23)00461-6 Publisher ID: 133746
                DOI: 10.1016/j.snb.2023.133746
                PMC ID: 10050199
                SO-VID: c454768f-468b-4363-b415-a248a358dec1
                Copyright © © 2023 Elsevier B.V. All rights reserved.
                License:

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                Date received : 27 January 2023
                Date revision received : 20 March 2023
                Date accepted : 27 March 2023
                Categories
                Subject: Article

                Keywords: sars-cov-2 s antigen,peptide,elisa,tyramine signal amplification,sars-cov-2 pseudoviruses
                Data availability:
                Keywords: sars-cov-2 s antigen, peptide, elisa, tyramine signal amplification, sars-cov-2 pseudoviruses

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