SARS-CoV-2 Spike Protein and Neutralizing Antibody Targeting the right antibody can have a major impact on understanding patient immune response.

Why use a SARS-CoV-2 antibody assay that targets the receptor-binding domain (RBD) of the S1 spike protein?

  • Cross-reactivity: Evidence showed an assay using the S1 RBD target did not cross-react with 4 common coronaviruses.
  • S1 RBD antibodies have shown strong correlation to neutralization while evidence for neutralizing antibodies to the Nucleocapsid (N) protein is sparse.
  • Potential vaccine response: The spike protein and particularly the RBD are the most common target of vaccine designs.
With hundreds of SARS-CoV-2 antibody tests on the market,1 healthcare providers (HCP) need to be confident that they can use results to provide actionable information to manage patient health. Assay design is proving to be a strong factor in how valuable different testing solutions are in helping HCP’s protect individual patients and communities.

Humans produce antibodies against the spike protein and S1 RBD, as well as other viral proteins, but the key questions to answer are “Are they neutralizing antibodies, and can they protect you from reinfection?” Multiple studies indicate a primary role for neutralizing antibodies that target the spike protein of SARS-CoV-2, with antibodies to the S1 RBD estimated to comprise ~90% of neutralizing activity.2-5

Many vaccines are targeting the RBD, therefore antibody assays that measure antibodies to the RBD will likely prove valuable to assess an individual’s immune response to the vaccine.

Deep dive into the spike protein and neutralization

A common part of the immune response is for humans to neutralize or block the binding of the virus to the ACE2 receptor. If an antibody interferes with binding, you may have some level of protection. Different antibodies will target different proteins in the viral structure.

Spike (S1 and S2) Protein:

The S1 protein is the head of the spike protein and contains a receptor-binding domain (RBD). The S1 RBD is instrumental for allowing the SARS-CoV-2 virus to reproduce by attaching to and infecting host cells.6

The S2 protein is the stalk of the spike protein and allows the SARS-CoV-2 virus to remain fused to host cells.6

Nucleocapsid (N) Protein:

  • The N protein is located inside of the capsid (the viral protein shell), along with RNA of SARS-CoV-2. This protein functions in the dispersal of genetic material during viral reproduction. The SARS-CoV-2 N protein is highly similar in structure to that of the SARS coronavirus (SARS-CoV).7
  • Tests that detect antibodies to the N and S proteins (including the S1 RBD antigen) have been developed and indicate an immune response to infection.

Data indicates the likelihood of cross-reactivity is impacted by the choice of target in SARS-CoV-2 assays and may be more likely in assays that target both S1 and S2 or the N protein versus assays targeting just S1 or S1 RBD.8

  • S1 RBD is highly specific for SARS-CoV-2 vs. other coronaviruses. Internal testing and an external study using the S1 RBD target showed that these assays did not cross react with the 4 very common coronaviruses.8
  • Evidence indicates that assays that target the N antigen may have cross reactivity with other coronaviruses, increasing the chances of false positive results and lower specificity.9

  • Testing directly for antibody associated with neutralization is important for accurate assessment of potential antibody-associated immunity.
    Granjean, L. Humoral response dynamics following infection with SARS-CoV-2. medRxiv. 2020. doi:
  • Neutralizing antibodies associated with spike (and particularly the RBD) can provide a level of protection/immunity. Use of tests that are associated with spike antibodies (such as S1 RBD) should be used to assess the population for prevalence studies
    Muecksch, F. Longitudinal analysis of clinical serology assay performance and neutralizing antibody levels in COVID19 convalescents. medRxiv. 2020. doi:
  • “Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.”
    McAndrew, K. Heterogeneous antibodies against SARS-CoV-2 spike protein receptor binding domain and nucleocapsid with implications on COVID-19 immunity. 2020. JCI Insigh. doi:
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