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Antibody Testing

Last Updated: October 5, 2020 

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The following is a curated review of key information and literature about this topic. It is not comprehensive of all data related to this subject.

Overview

Serologic tests for SARS-CoV-2 are antibody-based assays that measure an individual’s humoral immune response to SARS-CoV-2. Therefore, unlike nucleic acid amplification tests (NAATs, RT-PCRs), which detect viral RNA, serologic tests do not directly detect pathogens. A positive serology result may indicate either a current or past infection, and does not differentiate between the two. The tests currently available in the United States assess for binding antibodies, and generally detect IgG, IgM, or both IgG/IgM. These tests are now widely available, but offer variable sensitivity and specificity.  Commercial serologic tests to detect neutralizing antibodies are in development, but none have yet received FDA Emergency Use Authorization.   

Anti-SARS-CoV-2 antibodies typically become detectable approximately 2 or more weeks after the onset of symptoms (Guo, March 2020Zhao, March 2020; Compeer, August 2020). It seems both IgM and IgG rise around the same time (CDC, August 2020).  How long these antibodies stay detectable and their durability is not yet known (Korte, August 2020Vabret, July 2020). Serologic tests are therefore not useful early in the course of illness for diagnosing COVID-19. In addition, not all patients with SARS-CoV-2 infection develop an antibody response, and so a negative serologic result does not exclude past infection. Lastly, the sensitivity and specificity of tests are impacted by community prevalence.  

In order to have value as a diagnostic test, SARS-CoV-2 serologic tests should have a sensitivity and specificity of >= 99.5% (CDC, August 2020IDSA, August 2020). If a test for a disease has 90% sensitivity and 99% specificity, and the disease prevalence is 10%, the positive predictive value (PPV) is 90.9% and the negative predictive value (NPV) is 98.9% (in a population of 10,000 people, 990 tests will be positive, and 90 of those will be false). If the disease prevalence is 50%, the PPV will be 98.9% and the NPV will be 90.8% (in a population of 10,000 people, 4,500 tests will be positive, and 50 of those will be false) (CDC, August 2020).  

Specific situations in which serology testing in acute SARS-CoV-2 infection may be beneficial are outlined below. For example, some patients may experience continued manifestations of SARS-CoV-2 infection after several weeks of symptoms (Carfi, July 2020). If RT-PCR testing is negative in this scenario, serology testing may be helpful.  

Guidelines

IDSA guidelines do not recommend/suggest against using serologic testing to diagnose SARS-CoV-2 infection during the first 2 weeks (14 days) following symptom onset. 

  • When SARS-CoV-2 infection requires laboratory confirmation for clinical or epidemiological purposes, IDSA guidelines suggest testing for SARS-CoV-2 IgG or total antibody 3-4 weeks after symptom onset to detect evidence of past SARS-CoV-2 infection 
  • IDSA guidelines make no recommendation either for or against using IgM specific antibody tests to detect evidence of past SARS-CoV-2 infection. 
  • IDSA guidelines suggest using IgG antibody to provide evidence of COVID-19 infection in symptomatic patients with a high clinical suspicion and repeatedly negative NAAT testing. 
  • IDSA guidelines suggest against using IgM or IgG antibody combination tests to detect evidence of past SARS-CoV-2 infection. 
  • IDSA guidelines suggest using IgG antibody to provide evidence of COVID-19 infection symptomatic patients with a high clinical suspicion and repeatedly negative NAAT testing. 
  • In pediatric patients with multisystem inflammatory syndrome, IDSA guidelines suggest using both IgG antibody and NAAT to provide evidence of current or past COVID-19 infection. 

 

Key Literature

In summary: The performance characteristics of available SARS-CoV-2 serology tests are variable; some achieve excellent sensitivity and specificity, while others do not. In addition, the interpretation of these tests is highly dependent on community prevalence, the likelihood the patient who is being tested had COVID-19 and other individual patient characteristics. Currently these tests are most useful for seroprevalence studies and for specific clinical scenarios that are discussed below. SARS-CoV-2 IgG tests typically become detectable 2 or more weeks after infection; it is therefore not recommended to obtain these tests in the first 14 days of illness. Rather, if testing is desired (i.e., if the patient fits into one of the clinical scenarios outlined below), it should be obtained 3-4 weeks post symptom-onset. 

Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison (The National SARS-CoV-2 Serology Assay Evaluation Group, September 2020).

Study population:

  • Four SARS-CoV-2 antibody assays: SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay).
  • Sensitivity and specificity was derived from 976 pre-pandemic blood samples and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection (collected at least 20 days post symptom onset).

Primary endpoint:

  • To investigate the performance of 4 high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA.

Key findings:

  • The Abbott assay sensitivity was 92.7% (95% CI 90.2–94.8) and specificity was 99.9% (99.4–100%); the DiaSorin assay sensitivity was 95.0% (92.8–96.7) and specificity was 98.7% (97.7–99.3); the Oxford immunoassay sensitivity was 99.1% (97.8–99.7) and specificity was 99.0% (98.1–99.5); the Roche assay sensitivity was 97.2% (95.4–98.4) and specificity was 99.8% (99.3–100); and the Siemens assay sensitivity was 98.1% (96.6–99.1) and specificity was 99.9% (99.4–100%).

Limitations:

  • Limited by sample volumes, especially given the constraints imposed by dead volume requirements for liquid handling, and unable to do repeat analyses.
  • Subgroup analysis by timing of collection or disease severity was constrained by small numbers and larger studies would be of value.
  • The sample did not include children, and likely under-represented certain ethnic minority groups
  • Characteristics of the patient population from which the samples were derived were not , including characteristics that could affect serology results, such as immune compromised status

Overall, from four commercial SARS-CoV-2 antibody immunoassays tested, all assays achieved a sensitivity of at least 98% with thresholds optimized to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset. The authors note that even with such sensitivity and specificity, thousands of additional incorrect diagnoses can occur if millions of tests are done in large populations; at a 10% seroprevalence, the Siemens assay would generate an estimated 2800 total errors per million tests, while the DiaSorin assay at a rate of 16 700 total errors per million tests. Community prevalence of disease and individual patient characteristics must be taken into account when interpreting results.

Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset (Lou, May 2020).

Study population: 

  • 80 hospitalized patients with PCR-confirmed COVID-19 in China; 26 patients with severe COVID-19. 
  • The incubation period was 0-23 days with a median of 5 days (IQR, 2–10 days). 

Primary endpoint: 

  • Determine the impact of total antibody (Ab), IgM, and IgG levels in patients with COVID-19 using the Wantai SARS-CoV-2 Ab ELISA.  

Key findings: 

  • The seroconversion rates for Ab, IgM and IgG were 98.8%, 93.8% and 93.8%, respectively.  
  • Seroconversion time since exposure was significantly longer for patients with a long incubation period than for those with a short incubation period (21 d vs. 13 d, p<0.001). 
  • The first detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20 days post-exposure or 9, 10 and 12 days post-symptom onset, respectively.  
  • Antibody levels increased rapidly 6 days post-exposure, and were accompanied by a decline in viral load.  
  • In the second and third week of illness, the sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3%, respectively.  

Limitations: 

  • Asymptomatic patients were not included; therefore, these results are only generalizable to symptomatic patients. 
  • Most samples were collected 1 month post onset; therefore, the duration of antibodies cannot be estimated. 
  • The prevalence of COVID-19 in this community at the time was high, which may limit the generalizability of the test characteristics to other communities. 

Overall, in patients with COVID-19, serology testing provided an important complement to RNA testing in the later stages of illness. 

 

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) (Guo, March 2020). 

Study population: 

  • 208 plasma samples were collected from 82 confirmed and 58 probable cases (qPCR negative but with typical manifestation) of hospitalized patients with COVID-19 in Wuhan, China. 

Primary endpoint: 

  • Time kinetics of various antibodies produced against the SARS-CoV-2.  

Key findings: 

  • The median duration of IgM and IgA antibody detection was 5 (IQR, 3–6) days. 
  • IgG was detected 14 (IQR, 10–18) days after symptom onset. 
  • The positive rates were 85.4%(IgM), 92.7% (IgA), and 77.9% (IgG), respectively.  
  • In confirmed and probable cases, the positive rates of IgM antibodies were 75.6% and 93.1%, respectively.  
  • 22% (18/82) of the patients who were confirmed to be positive by qPCR were found to be negative by the IgM antibody tests. 13 of these patients were enrolled within less than 7 days after symptom onset. 
  • The positive detection rate was significantly increased (98.6%) when combining IgM ELISA assay with PCR for each patient compared with a single qPCR test (51.9%). 

Limitations: 

  • A cross-sectional sample of specimens was used to determine the kinetics of SARS-CoV-2 antibodies, and individuals may have differing kinetics for antibody development. 
  • The prevalence of COVID-19 in this community at the time of the study was high, which may limit the generalizability of the test characteristics to other communities. 
  • Whether patients were receiving potential immune modulating therapies, as were part of Chinese national guidelines at the time, is not stated; this may have affected antibody results. 

Overall, in this cross-sectional cohort study of patients with COVID-19, in confirmed and probable cases, the positive rates of IgM antibodies were 75.6% and 93.1%, respectively.  

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 (Zhao, March 2020).

Study population: 

  • 173 hospitalized patients with SARS-CoV-2 infection, confirmed by RT-PCR, in China; of whom 32 (18.5%) were critically ill. 

 Primary endpoint: 

  • Determine the impact of total antibody (Ab), IgM, and IgG levels in patients with COVID-19 using the Wantai SARS-CoV-2 Ab ELISA.  

Key findings: 

  • 535 plasma samples were collected from the 173 patients and tested for antibodies against SARS-CoV-2.  
  • The seroconversion rate for Ab, IgM and IgG was 93.1% (161/173), 82.7% (143/173) and 64.7% (112/173), respectively, with median seroconversion times of 11, 12 and 14 days, respectively. 
  • In the early phase of illness (within 7-day since onset), RT-PCR had the highest sensitivity of 66.7%, whereas the antibody assays had a positive rate of 38.3%. 
  • The presence of antibodies increased to 100% (Ab), 94.3% (IgM) and 79.8% (IgG) 15 days after the onset of symptoms. 
  • Combining RT-PCR and antibody tests significantly improved the sensitivity of the diagnosis for COVID-19 (p<0.001), even in early phase of 1-week since onset (p=0.007).  
  • A higher titer of Ab was independently associated with a worse clinical classification (p=0.006).  

Limitations: 

  • Most of the RT-PCR tests were from upper respiratory tract specimens, which are not as sensitive as using lower respiratory tract specimens. 
  • Samples were collected during the acute phase of infection. 
  • The prevalence of COVID-19 in this community at the time of the study was high, which may limit the generalizability of the test characteristics to other communities. 

Overall, in this case series of patients with COVID-19, the seroconversion rate during the acute phase of infection increased rapidly during the first 2 weeks. 

 

Additional Literature

Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection (Wang, Aug 2020). In this cohort study, the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies in 30 patients with COVID-19 were assessed.  SARS-CoV-2-specific NAb titers were low for the first 7–10 d after symptom onset and increased after 2–3 weeks. The median peak time for NAbs was 33 d (IQR 24–59 d) after symptom onset. NAb titers in 93·3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34·8% (IQR 19·6–42·4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0·41, p & 0·05).

SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness (Röltgen, August 2020). In this cohort study, 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals was assessed. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months. 

Viral dynamics and immune correlates of COVID-19 disease severity (Young, August 2020). In this prospective observational cohort study of 100 patients with confirmed SARS-CoV-2 infection, seroconversion occurred at a median of 12.5 days (IQR 9-18) for IgM and 15.0 days (IQR 12-20) for IgG; 54/62 patients (87.1%) sampled at day 14 or later seroconverted. Severe infections were associated with earlier seroconversion and higher peak IgM and IgG levels.   

Kinetics of viral load and antibody response in relation to COVID-19 severity (Wang, July 2020). In this small cohort study of 12 patients with severe COVID-19 and 11 mild patients, mild patients showed significantly lower IgM response. IgG responses were detected in most patients in both severe and mild groups at 9 days post onset and remained high level throughout the study. High-levels of neutralizing antibodies were induced after about 10 days post onset in both severe and mild patients, and were higher in the severe group. 

High neutralizing antibody titer in intensive care unit patients with COVID-19 (Liu, July 2020).  In this cohort study investigators compared the neutralizing antibody responses of eight COVID-19 patients admitted to the intensive care unit with those of 42 patients not admitted to the ICU. The peak serum geometric mean NAb titer was significantly higher among the eight ICU patients than the 42 non-ICU patients (7280 [95% CI 1468-36099]) vs (671 [95% CI, 368-1223]). The median number of days to reach the peak Nab titers after symptoms onset was shorter among the ICU patients (17.6) than that of the non-ICU patients (20.1). 

Antibody tests for identification of current and past infection with SARS-CoV-2 (Deeks, June 2020). A Cochrane Review examining the diagnostic accuracy of antibody tests in 57 publications determined combination of IgG/IgM had a sensitivity of 30.1% (95% CI 21.4 to 40.7) for 1 to 7 days, 72.2% (95% CI 63.5 to 79.5) for 8 to 14 days, 91.4% (95% CI 87.0 to 94.4) for 15 to 21 days. They also found there is insufficient data to estimate the sensitivity of serology 35 days or more post-symptom onset. 

Antibody Responses to SARS-CoV-2 at 8 Weeks Post infection in Asymptomatic Patients (Choe, June 2020).  In this small case series, the levels of SARS-CoV02 neutralizing antibodies were assessed in 8 asymptotic patients. Neutralizing antibody occurred in all 7 patients, and 5/7 (71%) had positive serologic testing.  

Antibody Detection and Dynamic Characteristics in Patients with COVID-19 (Xiang, April 2020). In this prospective cohort study of 85 hospitalized patients with confirmed COVID-19 and 24 patients with suspected COVID-19, sensitivity, specificity, PPV, and NPV, of IgM were 77.3%, 100%, 100%, 80.0%, respectively, and those of IgG were 83.3%, 95.0%, 94.8%, 83.8%. IgM and IgG seroconversion occurred as early as 4 days after symptom onset, but the majority of seroconversions occurred in the second week of illness. 

A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease (Huang, April 2020). In this systematic review of literature on antibody immunity to coronaviruses, including SARS-CoV-2 as well as the related SARS-CoV-1, MERS-CoV and human endemic coronaviruses, 5 areas of focus are reviewed: 1) antibody kinetics, 2) correlates of protection, 3) immunopathogenesis, 4) antigenic diversity and cross-reactivity, and 5) population seroprevalence.

 

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