Interim Guidelines for COVID-19 Antibody Testing

Interim Guidelines for COVID-19 Antibody Testing

Interim Guidelines for COVID-19 Antibody Testing in Clinical and Public Health Settings

Updated Dec. 16, 2022

Summary of Recent Changes

Key Points:

Antibody tests have public health value for monitoring and evaluating population levels of immunity, as well as clinical utility for patients.

  • Antibody tests that have received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA) may be used for both public health and clinical purposes. Individual performance characteristics for each test can be found in the test’s instructions for use (IFU).
  • Antibody testing should not be used to determine whether someone is currently infected with SARS-CoV-2. Viral tests detect current infection.
  • Antibody testing is not currently recommended to assess for immunity to SARS-CoV-2 following COVID-19 vaccination or to assess the need for vaccination in an unvaccinated person.

Introduction to COVID-19 Antibodies

When a person becomes infected with a pathogen, their immune system makes antibodies specifically to fight it. A person’s immune system can also safely learn to make antibodies through vaccination. Once you have antibodies to a particular disease, they provide some protection from that disease.

Even if a person does get sick, having antibodies can protect them from getting severely ill because their body has some experience in fighting that disease. How long this protection lasts can be different for each disease, each person, or influenced by other factors. Antibodies are just one part of a persons’ immune response.

Antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, can be detected in the blood of people who have recovered from COVID-19 or people who have been vaccinated against COVID-19. It is important to remember that some people with antibodies to SARS-CoV-2 may become infected after vaccination (vaccine breakthrough infection) or after recovering from a past infection (reinfection). For many diseases, including COVID-19, antibodies are expected to decrease or “wane” over time. As their antibodies wane, a person may become more vulnerable to SARS-CoV-2 infection. Even after a person’s antibodies wane, their immune system may have cells that remember the virus and that can act quickly to protect the person from severe illness if they become infected.

Antibody tests can detect the presence of these antibodies in serum within days to weeks following acute infection or vaccination. Antibody tests are not used for diagnosing a current case of COVID-19. Everyone, regardless of whether they have antibodies or not, should still take steps to protect themselves and others, including staying up to date on vaccination.

Recommendations for Use of Antibody Tests

Antibody tests can be used to:

  • Determine if a person has COVID-19 antibodies, which suggests past infection or vaccination.*
  • Aid in the diagnosis of multisystem inflammatory syndrome in children (MIS-C) and in adults (MIS-A).
  • Monitor and evaluate population levels of immunity.

*Antibody tests are not recommended or authorized by the FDA to assess someone’s immunity after COVID-19 vaccination or determine if they need to be vaccinated. However, EUA indications do not preclude use of antibody tests in vaccinated individuals in certain situations.

Antibody tests should not be used to:

  • Diagnose current infection.**
  • Determine if someone can return to work or school.
  • Group people together in settings such as schools, dormitories, and correctional facilities; or to exempt someone from screening testing.
  • Exempt a person who wears personal protective equipment (PPE) at work from following site-specific requirements

**Acute infection from SARS-CoV-2 is determined best by diagnostic testing using a nucleic acid amplification test (NAAT) or antigen test.

Choice of antibody test and testing strategy

For all clinical and public health purposes, it is recommended to use one of the numerous antibody tests for SARS-CoV-2 that have been authorized by FDA. The list of qualitative and semi-quantitative SARS-CoV-2 antibody tests granted an EUA by the FDA can be found on FDA’s website.

Antibody tests with very high sensitivity and specificity are preferred since they are more likely to exhibit high positive (probability that the person testing positive actually has antibodies) and negative predictive values (probability that the person testing negative actually does not have antibodies) when administered at least 3 weeks after the onset of illness.

Additional considerations when selecting an antibody test include:

  • IgG levels appear to decrease more slowly over time than levels of other classes of antibody. Therefore, assays that measure total antibody or IgG could have higher sensitivity than IgM assays as more time passes since a person’s last infection.
  • IgM antibody can persist for weeks to months following infection, though its persistence appears to be shorter than IgG’s; therefore, detection of IgM could suggest relatively recent infection.
  • Detection of persistent antibodies varies by the test used.

FDA has issued an EUA for surrogate neutralization tests, which are qualitative binding assays that detect antibodies that block the interaction between the virus and the cellular virus receptor (ACE-2) without using cells or infectious virus. Plaque reduction neutralization assays are considered the gold standard for detection of neutralizing antibodies, but require cells, infectious virus, and are difficult to standardize. Although the surrogate neutralization test exhibits correlation to a plaque reduction neutralization test, the clinical or public health applicability has not been established.

The clinical applicability of semi-quantitative tests has not been established.

Interpretation of anti-S and anti-N antibody results based on vaccination status

Vaccination status

Vaccination status

Vaccination status

Anti-S antibody

Anti-S antibody

Anti-S antibody

Anti-N antibody

Anti-N antibody

Anti-N antibody

Interpretation*

Interpretation*

Interpretation*

Vaccinated

Vaccination status

Vaccinated

+

Anti-S antibody

+

+

Anti-N antibody

+

Vaccinated and previously infected

Interpretation*

Vaccinated and previously infected

Vaccinated

Vaccination status

Vaccinated

+

Anti-S antibody

+

Anti-N antibody

Vaccinated and not previously infected

Interpretation*

Vaccinated and not previously infected

Unvaccinated

Vaccination status

Unvaccinated

+

Anti-S antibody

+

+

Anti-N antibody

+

Not vaccinated and previously infected

Interpretation*

Not vaccinated and previously infected

Unvaccinated

Vaccination status

Unvaccinated

Anti-S antibody

Anti-N antibody

Not previously vaccinated or infected

Interpretation*

Not previously vaccinated or infected

Unknown

Vaccination status

Unknown

+

Anti-S antibody

+

+

Anti-N antibody

+

Previously infected, may or may not have been vaccinated

Interpretation*

Previously infected, may or may not have been vaccinated

Unknown

Vaccination status

Unknown

+

Anti-S antibody

+

Anti-N antibody

Vaccinated with no previous infection

Interpretation*

Vaccinated with no previous infection

Unknown

Vaccination status

Unknown

Anti-S antibody

Anti-N antibody

Not previously vaccinated or infected

Interpretation*

Not previously vaccinated or infected

*Potential false positive or false negative results, failure to develop detectable antibodies after vaccination or infection, and waning of antibodies with time after infection or vaccination should be considered when interpreting antibody test results.

Antibody tests can be used in seroprevalence studies to estimate vaccine coverage, or immunity from infection or vaccination in a community.

A negative antibody test does not rule out previous infection. A proportion of persons who are infected with SARS-CoV-2 might not develop measurable antibodies, thereby limiting the sensitivity of any antibody test to detect previous infection in these individuals. Also, the extent to which seroreversion occurs varies according to the antibody test used.  In addition, measurable antibodies also can wane over time.

All eligible people should be vaccinated and stay up to date on vaccination, including unvaccinated people who have previously been infected and have detectable antibodies.

Indications for antibody testing

Antibody testing is not a replacement for virologic testing and should not be used to establish the presence or absence of acute SARS-CoV-2 infection.

Persons suspected of having COVID-19 who test positive by direct viral detection methods for SARS-CoV-2 (e.g., NAAT or antigen detection tests) typically begin to develop measurable antibody 7–14 days after illness onset, and by 3 weeks most persons will test positive for antibody. During this interval, the sensitivity of detecting infection using NAAT or antigen detection testing decreases and the sensitivity of serologic testing increases. Antibody testing may be useful to support the diagnosis of COVID-19 illness or complications of COVID-19 in the following situations:

  • A positive antibody test at least 7 days following acute illness onset in persons who had a previous negative antibody test (e.g., seroconversion) but did not receive a positive viral test might indicate SARS-CoV-2 infection between the dates of the negative and positive antibody tests.
  • A positive antibody test can help support a diagnosis when patients present with complications of COVID-19, such as multisystem inflammatory syndrome or other post-acute sequelae of COVID-19.
Antibody testing can be used for clinical and public health purposes to help differentiate antibodies produced due to past infection from those produced by vaccination by using tests that measure antibodies against different protein targets.

Although current EUA indications do not preclude the use of these tests in vaccinated individuals, none of the currently authorized tests have been specifically authorized to assess immunity or protection of people who have received a COVID-19 vaccine, including people with immunocompromising conditions.

Whether the test has been validated to specifically detect antibodies against the antigens employed by the test and whether the antigens cross-react with antibodies to antigens that are not employed by the test should be considered. The results of available anti-SARS-CoV-2 IgG antibody tests may be interpreted in the following way:

In a person never vaccinated:

  • Testing positive for antibody against N (nucleocapsid protein), S (spike protein), or RBD (receptor-binding domain of S protein) indicates prior infection.

In vaccinated people:

  • Testing positive for antibody against the vaccine antigen target, such as the S protein, while testing negative for other antigens (e.g., N) suggests that they have produced vaccine-induced antibody. They may have never been infected with SARS-CoV-2, or they may have had a previous infection, but the N protein antibodies have since waned.
  • Testing positive for antibodies other than the vaccine-induced antibody, such as the N protein, indicates resolving or past SARS-CoV-2 infection that could have occurred before or after vaccination.
  • Antibody testing is currently not recommended to assess for immunity to SARS-CoV-2 following COVID-19 vaccination.

Current vaccines distributed in the United States induce antibodies to S protein. The presence of antibodies to N protein indicates previous infection regardless of a person’s vaccination status, while presence of antibodies to S protein indicates either previous infection or vaccination. Since vaccines induce antibodies to specific viral protein targets, post-vaccination antibody test results will be negative in persons without a history of previous infection if the test used does not detect antibodies induced by the vaccine.

Antibody Testing in the United States

Antigenic targets

The choice of antigenic targets might help address different aspects of immune response. Antibody detection against receptor-binding domain (RBD) is considered to have higher correlation with functional aspects like ability to neutralize virus (6). Differential reactivity of S and N specific antibodies might be used to help differentiate previous infection from vaccination in serologic studies, particularly for vaccines that produce antibodies only against S protein (12540).

Multiple forms of S protein—full-length (S1+S2) or partial (S1 domain or RBD)—are used as antigens for antibody tests. S protein is essential for virus entry into cells and is present on the viral surface. Within the S protein, the RBD is more conserved than S1 or full-length S.

N protein is the most abundantly expressed immunodominant protein and is more conserved across coronaviruses than S.

Types of antibody testing

Different types of assays can be used to determine different aspects of the adaptive immune response and functionality of antibodies. The tests can be broadly classified to detect either binding or neutralizing antibodies.

Performance of antibody tests

Independently evaluated test performance and the approval status of tests are listed on an FDA website. Most authorized tests are qualitative (providing a result that is positive, negative, or indeterminate) or semi-quantitative (providing a numerical result using a scale that is unique to that assay and not comparable to other assays); however, authorized quantitative assays (providing a measured and scaled assessment of antibody levels) are also available.

FDA requires commercially marketed antibody tests for SARS-CoV-2 to receive Emergency Use Authorization (EUA) or approval. Multiple agencies—including FDA, the National Cancer Institute/National Institutes of Health (NCI/NIH), CDC, and the Biomedical Advanced Research and Development Authority (BARDA)—are collaborating with members of academia and the medical community to evaluate the performance of antibody tests independently using a well-characterized set of clinical specimens (serum and plasma) collected before and during the COVID-19 pandemic.

The World Health Organization has developed international standards for SARS-CoV-2 antibody tests that can serve as the foundation for the calibration of tests that quantify antibodies. Both laboratory and point-of-care antibody tests have received EUA from the FDA. Antibody testing technologies include single-use lateral flow tests where the presence of antibody is demonstrated by a color change on a paper strip (similar to a pregnancy test) and laboratory-based immunoassays that allow for processing of many specimens at the same time. The EUA letter of authorization includes the settings in which each test is authorized, based on FDA’s determination of appropriate settings for use during the public health emergency.

Development of Antibodies and Immunity

This section was last updated on January 24, 2022. It is no longer being updated but remains on this page for reference.

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