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    1. Diagnostics
    2. Hematology

    The significance of serology antibody testing for SARS-CoV-2

    June 24, 2020
    Red Blood Cells And Covid 19 4923341 5eeb944a75e73

    The World Health Organization (WHO) declared the coronavirus SARS-CoV-2 disease (COVID-19) outbreak a global pandemic on March 11, 2020.1 This is the third coronavirus to cross species, following the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012, and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1) in 2003.2 However, SARS-CoV-2 has had a more rapid spread with over 7.2 million confirmed cases and over 400,000 deaths worldwide.3 The United States has nearly 2 million confirmed COVID-19, cases and above 100,000 related deaths.3

    As of June 9, 2020, the U.S. Food and Drug Administration (FDA) had provided Emergency Use Authorizations (EUAs) for molecular-based detection and/or diagnosis of COVID-19 to 74 manufacturers and 36 high-complexity laboratories who had developed molecular-based laboratory developed tests.4 Also as of June 9, 2020, the FDA has issued only 19 EUAs for antibody-based detection tests. The FDA has not objected to the development and distribution of serology tests in identifying antibodies for SARS-CoV-2 as long as manufacturers apply for EUA within 10 days of notifying the agency of their intent to market the tests.5

    Why is serology testing important for SARS-CoV-2?

    Currently, SARS-CoV-2 infection is diagnosed by the detection of the virus via molecular-based techniques, such as real-time reverse transcriptase polymerase chain reaction (RT-PCR) or deep sequencing. However, these tests have a high rate of false negativity. 6-8 These detection systems rely on adequate viral load at the sample collection site.6 SARS-CoV-2 can cause both upper, but predominantly lower, respiratory tract infections. The positivity rate for the different samples ranges from 74.4 percent to 88.9 percent for sputum, 53.6 percent to 73.3 percent for nasal swabs, and a much lower rate in throat swabs collected ≥8 days post-disease onset.9

    The highest positivity rate has been shown in bronchoalveolar lavage fluid specimens (93 percent).10 However, lower respiratory tract sampling requires both a skilled technician and a suction device. Additionally, false-negative results are observed with RT-PCR methods when the viral replication time-window is missed.6 Liu et al. 2020 showed 11 days’ post symptom onset (PSO) is a transitional time point, when infected patients produce anti-viral antibodies, allowing confirmation of viral infection. The positivity rate for detection of IgM and/or IgG antibodies was >80 percent at this time point and was close to 100 percent after 14 days PSO. In contrast, the positivity rate of RT-PCR was about 60 percent 11 days PSO and began to rapidly decline afterwards (Figure 1).

    The overall positivity rate was significantly higher by antibody testing (81.5 percent) compared with RT-PCR (64.3 percent) at day 11 PSO.7 In another study, only 51.9 percent of patients were positive by a single RT-PCR test, but the positivity rate increased to 98.6 percent following and antibody assay on PCR- negative individuals.6 At this stage of the pandemic, a molecular false-negative result could pose a serious threat by allowing infectious patients back into the community, and hindering the efforts to contain virus spread.6 Therefore, a combination of RT-PCR and antibody testing methods would considerably improve diagnostic efficacy, even during early stages.7

    What is the significance of serology testing for SARS-CoV-2?

    Serological assays aid in understanding the immune responses to SARS-CoV-2 in a dynamic qualitative or quantitative manner and to identify individuals who were infected compared to those who were not. Data from serological tests are essential to determine the immune status of healthcare workers. Those who have developed the antibodies then can be deployed back to frontline work with less risk of illness while limiting virus spread.11 Serological assays are also needed to conduct epidemiologic studies to assess the extent of virus spread in communities and to determine the infection fatality rate. Furthermore, serological assays can identify individuals with the required antibody response against SARS-CoV-2, who can be donors of hyperimmune plasma to treat sick individuals.11,12 Lastly, serological assays can be used to select patients for clinical trials for vaccine or therapy development.2

    What are the target antigens for the serology testing for SARS-CoV-2?

    Figure 1. Positivity rate of patients based on detection of viral load by RT-PCR and antibodies by ELISA (Adapted from Liu et al. 2020.7).
    Figure 1. Positivity rate of patients based on detection of viral load by RT-PCR and antibodies by ELISA (Adapted from Liu et al. 2020.7).

    Coronavirus has four main structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). The S protein consists of the S1 and S2 subunits. The globular head S1 subunit contains the receptor binding domain (RBD) and facilitates the attachment to host cells, mediated by its interaction with cell surface angiotensin-converting enzyme 2 (ACE2).13 The S2 subunit comprises the stalk of the spike protein and facilitates fusion between viral envelope and host cell membrane after S1 binding induced conformational changes.14 The S protein is highly immunogenic since it is located on the surface of the virus.15

    The N protein plays an important role in the transcription and replication of viral RNA, packaging the encapsidated genome into virions16 and inhibits the cell cycle process of the host cells.17 The N protein is abundantly expressed during infections and also has high immunogenic activity.8,9,18 Therefore, both N and S protein could be potential targets for the antibody-based detection of SARS-CoV-2.9 However, the N protein homology between SARS-CoV-2 and SARSCoV-1 is 90 percent, compared with the S protein (77 percent), especially the S1 subunit including the RBD (66 percent).

    Additionally, the percentage of amino acid identity among other endemic coronaviruses (OC43, HKU1, 229E, NL63) and MERS-CoV was higher for the N protein, compared with the S1 subunit,2 suggesting that serodiagnostics using N protein could potentially demonstrate greater cross-reactivities among the endemic coronaviruses. Jiang et al. 2020 showed the N protein based antibody assays could exhibit a higher false-negative rate compared with the S1 subunit, and that S1 subunit purified from mammalian cells demonstrated the highest performance to distinguish COVID-19 patients from controls.19

    On the contrary, the N protein antigen is generally produced in bacteria instead of mammalian cell lines. Therefore, critical human glycosylation would be missing and, in turn, decrease high-affinity binding to antibodies.20 Therefore, the S1 subunit could be the specific target antigen for detecting SARS-CoV-2 antibodies.2

    What antibodies are detected against SARS-CoV-2?

    In infectious disease, IgM and IgG antibodies are commonly detected using serological tests due to their important role in tackling the viral infection. However, in many respiratory infections, in addition to IgM antibodies, IgA antibodies are produced in high titers during the early phase of the infection.21,22 In general, IgM antibodies have a lower affinity compared to IgA or IgG antibodies, and pose an increased risk of cross-reactivity against antigenically similar epitopes, which are common in coronaviruses.23

    During the SARS-CoV-1 infection of 2002, there was simultaneous seroconversion of all immunoglobulin classes (IgM, IgA and IgG),24,25 and similar observations are being reported for the SARS-CoV-2 infection. 6,20,26 Guo et al. 2020 showed that the production of IgA, IgM, and IgG antibodies was positive as early as one-day PSO. IgM/IgA antibodies and IgG antibodies were detected at a median of five and 14 days’ PSO, respectively.

    Additionally, during the acute phase (days 1-7, PSO), a higher positivity rate was observed for IgA antibodies (92.7 percent) compared with IgM antibodies (85.4 percent),6 indicating IgA antibodies could serve as a marker for acute infection. In another study, IgA antibodies were detected earlier than IgM antibodies, and the IgA values peaked at 20-22 days with persistently higher levels compared with IgM antibodies throughout the observation period (45 days PSO).

    In contrast, IgM levels peaked at 10-12 days and declined after 18 days.27 Jin et al. 2019 showed as the interval from RT-PCR confirmation to serological detection increased, the IgM positivity rate (75 percent) increased initially but subsequently declined (<50 percent). IgG positivity increased to 100 percent and remained stable thereafter. IgG levels were consistently higher than IgM levels,8 suggesting IgG antibodies persist longer in the body, and could contribute to long-term immune memory against SARS-CoV-2.28.

    Does the presence of antibodies indicate if someone is immune against SARS-CoV-2 reinfection?

    The presence of antibodies alone does not indicate that the person is immune against potential reinfection. It is important to determine whether the antibodies can confer protective immunity long-term, providing virus-neutralizing antibodies that block viral infection and help in clearing viral infection.14,28 Therefore, neutralizing antibodies play an important role in resolving viral infection. Plaque reduction neutralization tests (PRNT) in vitro are considered the gold standard for determining the ability of antibodies to neutralize virus and prevent viral replication.28 Neutralizing antibodies primarily target S protein in coronaviruses, in particular the S1 subunit and the RBD contained within the S1 domain, preventing viral entry into the host cell.29

    Studies from 2002 SARS-CoV-1 and 2012 MERS-CoV have reported neutralizing antibodies target the S protein, predominantly S1 or the RBD, and to a lesser extent S2,30 and similar observations are emerging in SARS-CoV-2. Neutralizing antibody responses against SARS-CoV-1 begin to develop by week two, and most patients develop neutralizing antibodies by week three.14 Okba et al. 2020 compared the performance of different ELISAs with PRNT assays to determine their ability to detect neutralizing antibodies to SARS-CoV-2. They found good correlation between antibody results in different ELISAs and the neutralizing test.

    The strongest correlation was observed with the EUROIMMUN IgA ELISA for infection reduction rates of >90 percent (PRNT90), suggesting the ability of EUROIMMUN ELISAs to detect SARS-CoV-2-specific antibodies.2 However, it is currently unknown whether the presence of antibodies to SARS-CoV-2 is sufficient to confer protective-immunity in vivo in infected individuals. SARS-CoV-2 is a novel pathogen, and there is no information regarding the duration or long-term effectiveness of these antibodies in recovered patients. SARS-CoV-1 specific antibodies became undetectable in 91 percent of patients after six years of infection,31 indicating longitudinal studies are required to further understand the potential role of SARS-CoV-2 specific antibodies in patients.

    Conclusion

    Detection of antibodies against SARS-CoV-2 virus complements viral testing. Antibody detection in combination with RT-PCR expands the detection window of SARS-CoV-2 infection and minimizes false-negative RT-PCR testing. It would provide valuable information to public health authorities making decisions to relax physical distancing measures and reopen businesses across the country. Antibody testing will help determine the extent of SARS-CoV-2 spread in communities. However, there are still questions that need to be answered such as how long would the antibodies last and if they are truly protective?28

    It is important to understand the antibody kinetics in SARS-CoV-2 patients. Studies have reported both IgA and IgM antibodies can be detected earlier than IgG antibodies.20,32 Additionally, IgA and IgM levels peak early post infection, but IgA is more robust and longer lasting. IgG levels peak at 21-25 days PSO.20 However, further studies are required to understand the long-term kinetics of these antibodies in infected individuals. Additionally, whether these antibodies can provide protection from reinfection and what antibody titers are required for protective immunity are yet to be determined. Furthermore, scientists are working on developing an effective vaccine, but the timeframe for its availability is unclear.

    With many states in the U.S. looking to re-open businesses, it is important to be vigilant and continue practicing social distancing and good hygiene practices until the role of antibodies against the SARS-CoV-2 virus is better understood.

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