Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 15;6(51):35657-35666.
doi: 10.1021/acsomega.1c05521. eCollection 2021 Dec 28.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen

Marketa Svobodova  1 , Vasso Skouridou  1 , Miriam Jauset-Rubio  1 , Irene Viéitez  2 , Alberto Fernández-Villar  3 , Jorge Julio Cabrera Alvargonzalez  4 , Eva Poveda  5 , Clara Benavent Bofill  6 , Teresa Sans  6 , Abdulaziz Bashammakh  7 , Abdulrahman O Alyoubi  7 , Ciara K O'Sullivan  1   8
Affiliations

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen

Marketa Svobodova et al. ACS Omega. .

Abstract

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Detection of the SARS-CoV-2 S antigen with the aptamer sandwich assay.
Figure 2
Figure 2
Specificity of the aptamers evaluated by direct ELAA. The relative binding of each aptamer was calculated using constant concentrations of aptamers (200 nM) and S1 proteins from the different HCoV’s (2 μg/mL). The error bars correspond to the standard deviation from duplicate measurements.
Figure 3
Figure 3
Screening of aptamer pairs for the detection of SARS-CoV-2 S1 protein. Each thiolated capture aptamer (AptxT) was analyzed in combination with each of the biotinylated reporter aptamers (AptxB) using a constant concentration of S1. No protein was added for the control samples. The number refers to the number of the aptamer tested, T indicates the thiolated aptamer and B indicates the biotinylated aptamer.
Figure 4
Figure 4
Sensitivity of the sandwich assays for SARS-CoV-2 S1 detection using different combinations of aptamer pairs.
Figure 5
Figure 5
Specificity of the sandwich assays using different aptamer pairs and S1 from different HCoV’s. Each protein was used at 2 μg/mL, and the error bars indicate the standard deviations from duplicate measurements.
Figure 6
Figure 6
Sensitivity of the sandwich assay based on the Apt1T/Apt5B aptamer pair in aptamer binding buffer and in VTM.
See this image and copyright information in PMC

References

    1. Weiss S. R. Forty years with coronaviruses. J. Exp. Med. 2020, 217, e2020053710.1084/jem.20200537. - DOI - PMC - PubMed
    1. He F.; Deng Y.; Li W. Coronavirus disease 2019: What we know?. J. Med. Virol. 2020, 92, 719–725. 10.1002/jmv.25766. - DOI - PMC - PubMed
    1. Zhu Z.; Lian X.; Su X.; Wu W.; Marraro G. A.; Zeng Y. From SARS and MERS to COVID-19: a brief summary and comparison of severe acute respiratory infections caused by three highly pathogenic human coronaviruses. Respir. Res. 2020, 21, 224.10.1186/s12931-020-01479-w. - DOI - PMC - PubMed
    1. Gorbalenya A. E.; Baker S. C.; Baric R. S.; de Groot R. J.; Drosten C.; Gulyaeva A. A.; Haagmans B. L.; Lauber C.; Leontovich A. M.; Neuman B. W.; Penzar D.; Perlman S.; Poon L. L. M.; Samborskiy D. V.; Sidorov I. A.; Sola I.; Ziebuhr J. The species Severe Acute Respiratory Syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020, 5, 536–544. 10.1038/s41564-020-0695-z. - DOI - PMC - PubMed
    1. Wrapp D.; Wang N.; Corbett K. S.; Goldsmith J. A.; Hsieh C.-L.; Abiona O.; Graham B. S.; McLellan J. S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020, 367, 1260–1263. 10.1126/science.abb2507. - DOI - PMC - PubMed