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SARS-CoV-2 Serology and PCR Testing

Accurate molecular tests for active SARS-CoV-2 infection and subsequent serological tests for the presence of anti-viral antibodies are key tools for fighting the COVID-19 pandemic. PCR-based testing is used to identify infected individuals, and serological testing is necessary to understand the immune response in those who have recovered. Development of specific and sensitive tests of both types (for diagnosis and antibody testing) is a critical need. 

Research, development and manufacturing of molecular tests and serology assays is a focus of many laboratories worldwide. Promega provides products and custom services to labs designing, performing and scaling up PCR-based molecular tests, conducting serology tests and researching new serological testing methods.

SARS-CoV-2 Targets for PCR and Serological Analysis

SARS-CoV-2 is a positive-sense, single-stranded RNA virus. The genome has been shown to share 79.6% identity with SARS-CoV, and the SARS-CoV-2 virus has been shown to bind to the same ACE2 cell surface receptor (1,2).  

The SARS-CoV-2 genome encodes multiple structural and non-structural proteins. Structural proteins include the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins (1,3). 

The structure of the viral spike protein was published in Science on February 19, 2020 (4). The spike protein has been shown to be the site of binding to the ACE2 receptor, and is of interest as a potential target for neutralizing antibodies (5,6) and as a target for therapies that could prevent attachment to target cells or internalization of the ACE2 receptor (7,8). Several viral proteins have also emerged as potential targets for the use of antiviral and other therapeutic agents (9).

SARS-CoV-2 structural proteins

SARS-CoV-2 Serologic Assay Development

PCR-based methods can only tell us if the virus is present in the patient at the time of sampling. They do not provide any information about past infection or the presence of anti-SARS-CoV-2 antibodies. Serological assays are needed to answer these important questions about individual exposure, to conduct population screening and surveillance, and ultimately to understand the adaptive immune response to this disease.

Considerable research effort is currently directed towards understanding the immune response to SARS-COV-2, including identifying viral antigens that elicit a protective response and developing serological tests to detect anti-SARS-CoV-2 antibodies.

The availability of specific and sensitive serological tests to demonstrate the presence of antibodies to the virus is vital to vaccine development efforts and to understanding whether an individual is safe from re-infection. Antibody tests need to be able to specifically detect antibodies against SARS-CoV-2 with no cross-reactivity to other coronaviruses.

SARS-CoV-2 and the Antibody Response in COVID-19 Patients

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“Many companies and research teams around the world are working to develop serological assays that can help us in the fight against COVID-19. Any test to be widely deployed must prove both sensitivity and specificity and must have the ability to determine if the antibody level is actually protective for the patient.”

- Ash Anderson, Promega Chief Medical Officer

Serological Assay Types

There are numerous COVID serology tests entering the market, and new papers published daily that contribute to the growing body of information on SARS-CoV-2 immune response. Studies on SARS-CoV and some preliminary studies on SARS-CoV-2 have suggested that nucleocapsid (N) and spike (S) proteins elicit antibody responses that may be protective, or be candidates for vaccine development (5-6,14-16). Many of the tests entering the market detect antibodies to N or S proteins (17). Several studies have demonstrated that antibodies produced against the receptor binding domain (RBD) of the spike protein are capable of viral neutralization (18-23). Studies are ongoing to answer many remaining questions about immunity to SARS-CoV-2, including whether IgG antibodies developed to the virus are protective over the long term.

Conventional ELISA and chemiluminescent immunoassays (CMIA), and point-of-care (POC) lateral flow tests are the main types of SARS-CoV-2 serological test currently available. The Lumit™ Dx SARS-CoV-2 Immunoassay, a novel detection method based on bioluminescence technology, provides advantages of speed and scalability when compared to ELISA and lateral flow methods.

Lumit? Technology-Based Test

The Lumit™ Dx SARS-CoV-2 Immunoassay uses a SARS-CoV-2-specific protein (spike protein) labeled with two subunits of NanoBiT® technology (Lumit™ Dx CoV-2-SmBiT and Lumit™ Dx CoV-2-LgBiT). When the labeled proteins are incubated with a sample containing SARS-CoV-2 antibodies, the proteins bind to the antibody, bringing the SmBiT and LgBiT subunits into close proximity to form a functional luciferase. A bright luminescent signal is generated following the addition of the Lumit™ Dx Detection Reagent, and the signal is detected using a microplate reader.

Unlike conventional ELISA methods, the Lumit™ Dx SARS-CoV-2 Immunoassay requires no wash steps, allowing faster, more reliable sample processing. The assay is performed in 96-well plates and generates results in 1 hour or less.

This product is currently only available in the US.

View Product

ELISA-Type Tests

Numerous ELISA or chemiluminescent immunoassays are on the market that use viral antigens to capture antibodies in test samples, many using SARS-CoV-2 spike or nucleocapsid proteins as targets. ELISA-type tests offer the advantage of quantifying antibody responses, but take longer to perform than point-of-care lateral flow-type tests as they involve more reagent addition and wash steps. 

Here is a video explaining ELISA test methods.

Lateral Flow Tests

In a lateral flow test, the sample is added to a solid support strip containing viral antigens labeled with colloidal gold, and with anti-human IgM and IgG located in specific sections of the strip. Anti-SARS-CoV-2 antibodies present in the sample bind the viral antigens and are then captured by the anti-human antibodies as they travel across the test strip. A visible line of colloidal gold appears, indicating the presence of IgM or IgG antibodies in the test sample.

Here is a video explaining lateral flow tests.

PCR-Based SARS-CoV-2 Testing Methods

RT-qPCR based assays designed to amplify SARS-CoV-2-specific sequences are the primary method currently used for detection of active infections. Tests are available targeting various unique SARS-CoV-2 sequences in the N, E, S and RdRp sequences (3,10). RT-qPCR tests that use two or more targets are likely to have higher specificity. 

The World Health Organization (WHO) has published summary information for a few protocols including the United States recommended protocol (11). The CDC recommends using the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for primary diagnosis, which includes primers and probes for different regions of the nucleocapsid (N) gene plus an additional primer/probe set for detection of RNase P (RP) gene in control/clinical samples (12,13).

Other organizations have released their own kits. On February 25 2020, Promega was recognized by Co-Diagnostics, Inc. for support in manufacturing the new Logix Smart™ COVID-19 Test, which received CE mark approval and is available in Europe as an in vitro diagnostic.

“Promega is proud to support the CDC’s rapid response efforts and COVID-19 testing labs with approved  amplification technology"
Heather Tomlinson, Promega Director of Clinical Diagnostics

Product Option for CDC COVID-19 Diagnostic Panel Protocol

The GoTaq® Probe 1-Step RT-qPCR System is a recommended master mix option in the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel that is available through the FDA’s Emergency Use Authorization (EUA).

Preparing Samples for Direct PCR

XpressAmp™ Direct Amplification Reagents enable direct PCR without prior RNA or DNA extraction. These reagents are used to prepare viral samples for analysis in qPCR or RT-qPCR assays.

The CDC 2019-Novel Coronavirus (2019- nCoV) Real-Time RT-PCR Diagnostic Panel is a test protocol for detection of SARS-CoV- 2 unique sequences in respiratory specimens.

View Protocol

References

  1. Zhou, P.  et al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-3.
  2. Zhu, N. et al. (2020) A Novel Coronavirus from Patients with Pneumonia in China, 2019NEJM 382:, 727-33.
  3. Li, X. et al. (2020) Molecular immune pathogenesis and diagnosis of COVID-19. J. Pharm. Anal.10, Issue 2, (April) 102-8.
  4. Wrapp, D. et al. (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367, 1260-3.  
  5. Walls, A.C. et al. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Cell 181(2), 281-292.e6.
  6. Wang, C. et al.  (2020) A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun 11, 2251.
  7. Richardson, P. et al. (2020) Baricitinib as potential treatment for 2019-nCoV acute respiratory disease. Lancet 395, e30–e31 (2020).
  8. Hoffmann, M. et al. (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181(2), 271-280.e8.
  9. Wu, R., et al. (2020) An Update on Current Therapeutic Drugs Treating COVID-19. Curr Pharmacol. Rep. May 11; 1‐15. doi:10.1007/s40495-020-00216-7. 
  10. Corman, V.M. et al. (2020) Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill., 25, 10.2807/1560-7917.ES.2020.25.3.2000045.
  11. World Heath Organization PCR protocol - World Health Organization. Accessed on May 13, 2020. https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2.
  12. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Accessed May 13, 2020. https://www.fda.gov/media/134922/download.

  13. Coronavirus Disease 2019 (COVID-19): FAQ for laboratories Accessed May 13, 2020. https://www.cdc.gov/coronavirus/2019-ncov/lab/testing-laboratories.html.

  14. Tian, C. et al. (2020) Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibodyEmerg. Microb. Infect., 9, 382-85.
  15. Ahmed, S.F. et al. (2020) Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses 12(3), 254. 
  16. Tay, M.Z., et al. (2020) The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol.  https://doi.org/10.1038/s41577-020-0311-8.
  17. Serology-based tests for COVID-19. Johns Hopkins University Center for Health Security.
  18. Premkumar, L., et al. (2020) The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol5, doi:10.1126/sciimmunol.abc8413 (2020).
  19. Ju, B., et al. (2020) Human neutralizing antibodies elicited by SARS-CoV-2 infectionNature, doi:10.1038/s41586-020-2380-z (2020).
  20. Shi, R., et al. (2020) A human neutralizing antibody targets the receptor binding site of SARS-CoV-2Nature, doi:10.1038/s41586-020-2381-y (2020).
  21. Rogers, T. F., et al. (2020) Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal modelScience (New York, N.Y.), doi:10.1126/science.abc7520 (2020).
  22. Seydoux, E., et al. (2020) Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic MutationImmunity, doi:10.1016/j.immuni.2020.06.001 (2020).
  23. Brouwer, P. J. M., et al. (2020) Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerabilityScience (New York, N.Y.), doi:10.1126/science.abc5902 (2020).
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