Using Antibody Testing in Clinical Practice
How can the results of antibody testing and viral diagnostic testing be used to provide additional detail on a patient's clinical presentation?
- The White House has issued Guidance on the interpretation of COVID-19 Test Results to assist with making patient care decisions and recommending protective measures based on scenarios of positive and negative antibody and viral testing results.
What directives or recommendations are being provided regarding use of serologic (antibody) testing in clinical practice?
- Guidelines, public health policies, and clinical recommendations are subject to frequent changes as more information becomes available about the immune response to SARS-CoV-2 and the role of antibody testing. Frequent review of this information is recommended to most appropriately utilize antibody testing in any given clinical or public health setting.
- The Infectious Disease Society of America (IDSA) released clinical practice guidelines on August 18, 2020. An expert panel was convened to perform a systematic review of the coronavirus disease 2019 (COVID-19) serology literature and construct best practice guidance related to SARS-CoV-2 serologic testing.
- The CDC has also provided Interim Guidelines for COVID-19 Antibody Testing which are continually updated on their site as new information becomes available. Highlights of their recommendations can be summarized as follows:
- Serologic assays that have Emergency Use Authorization (EUA) are preferred for public health or clinical use since their test performance data have been reviewed by FDA.
- Assays with high specificity are recommended to reduce false-positive results and testing should be performed on individuals and populations with an elevated likelihood of previous exposure to SARS-CoV-2.
- Recommendations regarding utilizing highly specific assays are echoed by the IDSA.
- Currently, there is no identified advantage whether the assays test for IgG, IgM and IgG, or total antibody.
- There are some variabilities of sensitivity and specificity among the antibody types that have been described in literature, and the IDSA has summarized those differences in data tables viewable here.
- Serologic testing should not be used to determine immune status in individuals until the presence, durability, and duration of immunity are established.
- Serologic testing can be offered as a method to support diagnosis of acute COVID-19 illness for persons who present late. For persons who present 9-14 days after illness onset, serologic testing can be offered in addition to recommended direct detection methods such as polymerase chain reaction. This will maximize sensitivity as the sensitivity of nucleic acid detection is decreasing and serologic testing is increasing during this time period. (CDC)
- The IDSA provides data comparing nucleic acid amplification testing (NAAT) alone vs. NAAT plus serology (when initial NAAT is negative) for diagnosing COVID-19 in symptomatic patients.
- Serologic testing should be offered as a method to help establish a diagnosis when patients present with late complications of COVID-19 illness, such as multisystem inflammatory syndrome in children. (CDC)
- The IDSA specifically suggests using both NAAT and IgG antibody testing in this scenario.
- Serologic testing by itself should not be used to establish the presence or absence of SARS-CoV-2 infection or reinfection. Antibodies may not be present among those tested early in illness before antibodies develop or among those who never develop detectable antibodies following infection. In addition, the presence of antibodies may reflect previous infection and may be unrelated to the current illness.
- The IDSA provides data showing antibody performance within the first two weeks of illness.
- The FDA recommends health care providers:
- Continue to use serological (antibody) tests, as appropriate, and be aware of their limitations. Serologic testing should not be used to determine immune status in individuals until the presence, durability, and duration of immunity is established.
- Do not use serological (antibody) tests as the sole basis to diagnose COVID-19 but instead as information about whether a person may have been exposed.
- According to the CDC, it typically takes 1 to 3 weeks after someone becomes infected with SARS-CoV-2 for their body to make antibodies; some people may take longer to develop antibodies.
- Although serologic tests should not be used at this time to determine if an individual is immune, these tests can help determine the proportion of a population previously infected with SARS-CoV-2 and provide information about populations that may be immune and potentially protected. Thus, demographic and geographic patterns of serologic test results can help determine which communities may have experienced a higher infection rate and therefore may have higher rates of herd immunity.
- Some states and organizations are implementing antibody testing provisions in their policies to help guide COVID-19 diagnostic testing. Nursing homes and other institutions with COVID-19 testing requirements should inquire with their state department of public health to learn more about how antibody testing can be incorporated into these testing programs. Example Policies:
- Tennessee – Long-Term Care Facility Testing
- "Any staff member who has a positive FDA-approved COVID-19 antibody test is exempted from weekly testing."
How do I decide if a patient needs to be tested for SARS-CoV-2 antibodies?
- Many countries are now testing for SARS-CoV-2 antibodies at the population level or in specific groups, such as health workers, close contacts of known cases, or within households. The WHO describes some of these epidemiological efforts in detail. Serology testing can assist with contact tracing and understanding the spread of SARS-CoV-2 within specific populations and settings. Serology data will be vital in the coming months to optimize ongoing public health strategies and vaccine development.
- Providers considering SARS-CoV-2 antibody testing may consider the following in informing their decision to test:
- History of positive SARS-CoV-2 diagnostic test
- History of symptoms consistent with COVID-19 illness in the past (e.g., fever, cough, shortness of breath, loss of taste/smell)
- Individuals with an elevated likelihood of previous exposure, such as:
- Occupational exposure (e.g. healthcare workers, sanitation workers, etc.)
- Caregiver exposure (e.g. home aide, direct care provider in home, etc.)
- Close contact exposure (e.g., member of household diagnosed with COVID-19)
- Residential exposure (e.g., nursing home, homeless shelter, prison, group home)
- Recent travel to/from areas with higher numbers of COVID-19 cases
If patients have positive SARS-CoV-2 antibody test results, are they safer or better suited to return to work, volunteer in the community, or engage in activities that may put them in contact with persons infected with COVID-19?
- According to the CDC serologic test results should not be used to make decisions about grouping persons residing in or being admitted to congregate settings, such as schools, dormitories, or correctional facilities.
- Serologic test results should not be used to make decisions about returning persons to the workplace.
- According to the World Health Organization, there is currently no evidence that people who have recovered from COVID-19 and have antibodies are protected from a second infection.
Antibody Testing Methodologies and Specimen Types
What types of antibody testing are available? What specimen types can be utilized for antibody testing?
- The CDC describes several different types of assays that can be used to determine different aspects of immune response and functionality of antibodies. The tests can be broadly classified to detect either binding or neutralizing antibodies.
- Binding antibody detection: These tests use purified proteins of SARS-CoV-2, not live virus, and can be performed in lower biosafety level laboratories (e.g., BSL-2). With specific reagents, individual antibody types, like IgG, IgM, and IgA, can be determined. Depending on the complexity of assays, these tests can be performed rapidly (less than 30 minutes) in a field setting or in a few hours in a laboratory. Tests that detect binding antibodies fall into two broad categories.
- Neutralizing antibody detection:The FDA has not yet authorized the use of neutralization tests for SARS-CoV-2. Neutralization tests determine the functional ability of antibodies to prevent infection of virus in vitro. The test involves incubating serum or plasma with live virus followed by infection and incubation of cells. Testing will require either BSL-3 or BSL-2 laboratories, depending on what form of the SARS-CoV-2 virus is used. Two types of neutralization tests are conducted.
- Virus neutralization tests (VNT), such as the plaque-reduction neutralization test (PRNT) and microneutralization, use a SARS-CoV-2 virus from a clinical isolate or recombinant SARS-CoV-2 expressing reporter proteins. This testing requires BSL-3 laboratories and may take up to 5 days to complete.
- Pseudovirus neutralization tests (pVNT) use recombinant pseudoviruses (like vesicular stomatitis virus, VSV) that incorporate the S protein of SARS-CoV-2. This testing can be performed in BSL-2 laboratories depending on the VSV strain used.
- Specimen Types Utilized:
- Several of the currently authorized antibody tests can use serum or plasma samples (with certain specifications provided in the instructions for use). There are also approved tests that can use whole blood or a dry blood spot. Please refer to the instructions for use for the particular test in question to determine what specimen types are appropriate.
Considerations for each specimen type:
Refer to the instructions for use and sample collection and handling instructions provided by the laboratory.
Venipuncture required (phlebotomist)
Must be free of fibrin, red blood cells, and particulates
Bubbles can affect analysis
Storage in freezer may be necessary
May be required to ship frozen with cold pack
Multiple freeze/thaw cycles may contribute to degradation
Venipuncture required (phlebotomist)
Cannot be hemolyzed
Tests validated with whole blood may not be able to use finger stick blood (check instructions for use)
Stability window may be short (<24 hours)
Dry Blood Spot
Minimally invasive, small sample volume
Potential for self-sampling
No centrifugation or separation required before shipment
Ease of shipment (no cold chain or special handling)
Stable for several weeks at room temperature
Reduced biohazard risk for transit and lab personnel
McDade TW, McNally EM, D'Aquila R, et al. Enzyme immunoassay for SARS-CoV-2 antibodies in dried blood spot samples: A minimally-invasive approach to facilitate community and population-based screening. https://doi.org/10.1101/2020.04.28.20081844
What is the difference between immunoglobulin M (IgM) and immunoglobulin G (IgG), and how are these immune markers expressed over the course of a COVID-19 infection and recovery?
- According to the CDC, nearly all immune competent individuals will develop an immune response following SARS-CoV-2 infection. Like infections with other pathogens, SARS-CoV-2 infection elicits development of IgM and IgG antibodies, which are the most useful for assessing antibody response because little is known about IgA response in the blood. Antibodies in some persons can be detected within the first week of illness onset. SARS-CoV-2 infections are somewhat unusual because IgM and IgG antibodies arise nearly simultaneously in serum within 2 to 3 weeks after illness onset. Thus, detection of IgM without IgG is uncommon. How long IgM and IgG antibodies remain detectable following infection is not known.
Are there antibody tests for SARS-CoV-2 that have been approved by the FDA for use at the point-of-care?
- Several tests have been approved to provide rapid results for IgM/IgG antibodies. Some of these tests utilize lateral flow technology, and results are designed to be read within 20 minutes in most cases. Collection by a healthcare professional is required and is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C 263a, to perform moderate or high complexity tests. Due to the risk of false positive results, confirmation of positive results should be considered using a second, different IgG or IgM assay. The FDA's EUA page for serology tests is the most current resource for this information. It is unclear where these tests may be available and are being administered. One significant limitation of these tests is their low throughput. Only a limited number of tests per day could be performed using these point-of-care methods due to the individualized effort required for each test. Aegis Sciences Corporation is capable of processing up to 10,000 samples per day with a reliable turnaround time.
Aegis SARS-CoV-2 Antibody Testing Method, Performance Characteristics, and EUA Information
Can you provide specifics regarding the SARS-CoV-2 antibody testing provided by Aegis Sciences Corporation?
- Aegis offers a dry blood spot assay for determining presence of IgG antibodies utilizing the PerkinElmer GSP®/DELFIA® kit. This is a fluoroimmunoassay method targeting the spike protein. The method utilizes an automatic puncher to place the samples into plates, ensuring consistent sampling for testing and easy identification of the sample throughout the testing process.
- According to the CDC, testing for IgG may be a better marker of durable immunity to SARS-CoV-2. However, additional data is needed on the maturation of the immune response to SARS-CoV-2 to make evidence-based recommendations for all testing scenarios.
What is the specificity/sensitivity of the Aegis SARS-CoV-2 antibody test?
- Sensitivity: 96.1%
- Specificity: 99.9%
Note: The IDSA and CDC both state that choosing a test with high specificity is especially important to reduce false positive results.
Aegis Sciences Corporation also performed an internal validation study and noted similar performance characteristics.
No false positive results were observed in the internal validation study.
Similar to the manufacturer's study, sensitivity is greatly increased when testing patients >20 days after symptom onset or positive PCR result. Testing sooner than this increases the risk of false-negative results.
Negative results do not preclude acute SARS-CoV-2 infection. If acute infection is suspected, direct testing for SARS-CoV-2 is necessary. Results from antibody testing should not be used to diagnose or exclude acute SARS-CoV-2 infection.
Can positive results be due to other vaccines or viruses?
- The assay used by Aegis targets the S1 subunit of the spike protein. Within the coronavirus family, the S1 protein does not share much homology between the human pathogenic members of the family. An exception to this is the similarities between SARS-CoV-1 and SARS-CoV-2. Cross reactivity may occur with SARS-CoV-1 IgG response, but not with the IgG response caused by the other human coronaviruses.
- According to the CDC, SARS-CoV-1 did not spread widely in the community in the United States. A little over 8,000 people worldwide became sick with SARS during the 2003 outbreak, however in the U.S., only eight people had laboratory evidence of SARS-CoV infection. Based on this epidemiologic data, false positives due to SARS-CoV-1 antibodies would be extremely rare.
- On rare occasions, positive results may be due to past or present infection with non-SARS-CoV-2 coronavirus strains, such as coronavirus HKU1, NL&3, OC43 or 229E.
- People around the world commonly get infected with human coronaviruses, which were first identified in the 1960s. There are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta. The seven coronaviruses that can infect people are:
- 229E (alpha coronavirus) - common
- NL63 (alpha coronavirus) - common
- OC43 (beta coronavirus) - common
- HKU1 (beta coronavirus) - common
- MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS)
- SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS)
- SARS-CoV-2 (the novel coronavirus that causes COVID-19)
- Sometimes coronaviruses that infect animals can evolve and make people sick and become a new human coronavirus. Three recent examples of this are 2019-nCoV (or SARS-CoV-2), SARS-CoV, and MERS-CoV.
How is Aegis authorized to complete antibody testing for SARS-CoV-2?
- The authorization process for antibody testing is similar to the molecular diagnostic emergency use authorization (EUA) process. The process has been designed to allow laboratories with validated methods to go ahead and provide testing while awaiting EUA.
- Under the FDA's March 16 policy for serological tests, the FDA provided regulatory flexibility for developers offering such tests without FDA review and without an EUA where they have notified FDA that they have validated their tests and provide disclaimers about the limitations of the tests with any results generated by their tests, as outlined in the policy.
- FDA's policy was based on the considerations that serology tests are not meant to diagnose active SARS-CoV-2 infection and that early availability and use of these tests could help answer critical questions about the prevalence of COVID-19 infections in different communities, whether the presence of antibodies conveys immunity, and, if so, for how long.
- Serology tests that have received emergency use authorization from the FDA are listed here. Be aware that not all marketed serological tests have been evaluated by the FDA. The FDA's authorized tests, including serological tests, are listed on the EUA page. Tests being offered under a policy outlined in the FDA's COVID-19 Diagnostic Policy Guidance are listed on the FDA's FAQ page. Such tests have not been reviewed by the FDA, unless an EUA has also been submitted and reviewed by FDA. The CDC also states that serologic assays that have EUA are preferred for public health or clinical use since their test performance data have been reviewed by FDA.
Test Timing & Result Interpretation
Has there been any guidance regarding the "best" time to test for SARS-CoV-2 antibodies?
- The CDC reminds that antibody test results should not be used to diagnose someone with an active SARS-CoV-2 infection. It typically takes 1 to 3 weeks after someone becomes infected with SARS-CoV-2 for their body to make antibodies; some people may take longer to develop antibodies. Depending on when someone was infected and the timing of the test, the test may not find antibodies in someone with an active infection. A test that detects the SARS-CoV-2 virus, such as one that checks respiratory samples, should be used to test for active infection.
What evidence is available regarding timing of onset of COVID-19 symptoms, positive diagnostic test, and positive antibody test results? Have any case reports been studied for the time course of these events?
- The Sanford Guide references a Mt. Sinai study of 624 New York City patients with mild disease which found that IgG antibodies develop over a period of 7 to 50 days from symptom onset and 5 to 49 from symptom resolution, with a median of 24 days from symptom onset to higher antibody titers, and a median of 15 days from symptom resolution to higher antibody titers. All but 3 (0.5%) subjects with PCR-confirmed infections seroconverted; optimal time frame for widespread antibody testing is at least three to four weeks after symptom onset and at least two weeks after symptom resolution.
- Based on the results of the validation study conducted by PerkinElmer, the manufacturer of the test offered by Aegis, the sensitivity of the test greatly increased >20 days after a positive RT-PCR diagnostic test or symptom onset. The chance of false negative results is reduced if testing >20 days after a positive RT-PCR diagnostic test or onset of symptoms.
- Studies are ongoing to gauge the timing of symptom onset and immune response over the disease course. Aegis is reviewing published guidance for any updates or formal recommendations regarding the optimal timing of antibody testing.
How should the results of antibody testing be interpreted?
- What does it mean if a patient has a positive antibody test result?
According to the CDC, when antibodies are found (a positive test result), it means that a person was infected with SARS-CoV-2 and their body's immune system responded to the virus at some point in the past. People develop antibodies when their body's immune system responds to an infection. These antibodies can be found in the blood of people previously infected whether or not they had signs or symptoms of illness. The CDC offers these recommendations for persons who test positive for anti-SARS-CoV-2 antibodies
Although the presence of anti-SARS-CoV-2 antibodies when detected using a testing algorithm with high positive predictive value for the context of use likely indicates at least some degree of immunity, until the durability and duration of immunity is established, it cannot be assumed that individuals with truly positive antibody test results are protected from future infection.
Asymptomatic persons who test positive by serologic testing and who are without recent history of a COVID-19 compatible illness have a low likelihood of active infection and should follow general recommendations to prevent infection with SARS-CoV-2 and otherwise continue with normal activities, including work.
Persons who have had a COVID-19-compatible or confirmed illness should follow previous guidance regarding resumption of normal activities, including work.
There should be no change in clinical practice or use of personal protective equipment (PPE) by health care workers and first responders who test positive for SARS-CoV-2 antibody.
The CDC reports that scientists do not know if the antibodies that result from SARS-CoV-2 infection will provide someone with immunity from a future infection. If antibodies do provide immunity, we don't know what titer or amount of antibodies would be protective or the duration that protection would last. CDC scientists are conducting studies to better understand the level of antibodies needed for protection, the duration of that protection, and the factors associated with whether a person develops a protective antibody response.
Could a patient who is positive for SARS-CoV-2 antibodies still be actively shedding virus?
Yes. Positive antibody test results are neither diagnostic nor indicative of clearance or resolution of the SARS-CoV-2 infection. The CDC guidelines for discontinuing isolation in healthcare and non-healthcare settings should be utilized in conjunction with diagnostic (RT-PCR) testing and local public health recommendations to assess a person's readiness to discontinue isolation.
Is antibody testing for SARS-CoV-2 part of the assessment process to determine if a patient has cleared the infection?
Currently, no. More information is needed to determine the timing of antibody production relative to changes in viral RNA levels. At this time, patients may be evaluated for appropriate discontinuation of isolation according to the strategies described by the CDC (for persons not in healthcare settings and patients in healthcare settings) and in consultation with local public health officials.
What does it mean if a patient has a negative antibody test result?
Negative serology test results have more than one meaning. When antibodies are not detected, it could mean:
The person was not infected with SARS-CoV-2.
Infection occurred, but the body's immune response was not strong enough to make enough antibodies.
There may not have been enough time for antibodies to develop.
What factors can contribute to a positive RT-PCR diagnostic test and a negative antibody test, or vice versa?
Timing of testing is important. If an RT-PCR test is conducted too early or too late in the disease course, the viral load may be below the limit of detection. If an antibody test is conducted too early, the patient may not have undergone seroconversion and the result could be a false negative. The duration of IgG response is unknown, so at this time, it is not known if there is a point that could be "too late" for antibody testing.
Test performance characteristics greatly impact the validity of the results. If sensitivity and specificity are low for a diagnostic or antibody test, the associated risk of false negatives and false positives should be considered.
Proper collection impacts results. As discussed on our diagnostic testing FAQ page, proper collection of a nasopharyngeal, oropharyngeal, or nasopharyngeal specimen is essential to avoid false negative results. Insufficient sample collection can contribute to false negative results. When collecting a dry blood spot sample for antibody testing, it is essential to spot all five circles on the collection card to avoid test cancellation or false negative results.
If different laboratories performed the diagnostic and antibody testing, consider that testing methods differ between laboratories. It is recommended to contact the testing laboratory to find out specific information about the testing performed and relevant considerations in result interpretation.
What can be done to assist the drying process of the blood spot cards after collection?
- The samples should be allowed to air dry on a horizontally level, nonabsorbent, open surface for at least 3 hours at an ambient temperature (18-25° C). The cards should not be placed in front of air vents or other sources of moving air. Cards should be kept away from direct sunlight, and heat should not be used.
What information must be completed on the requisition form to ensure a sample is not rejected for testing due to missing information?
- Indicate the proper diagnosis code for the patient's clinical condition.
- Supply patient insurance information.
- Select an ordering provider.
- Fill in the date the sample is collected.
- Fill in the patient's name and date of birth on the label located at the top of the requisition form.
- Peel off the label and place lengthwise on the blood collection card, just below the blood spot circles.
- Complete the patient information section on the right hand side of the requisition form in its entirety.
- Obtain signatures from the ordering provider and patient.
- Electronic ordering is available. Please discuss with your Regional Sales Manager.
How will Aegis be reporting results?
- Results are reported qualitatively as positive or negative for IgG.
- Aegis is required to report results to the individual state's department of health.
- Ordering providers may receive results via the secure client web portal. E-mail notifications regarding results can be set up through the portal. Results may also be received via fax.
- Patients may receive their results via the secure online patient portal. Patients are asked to please check the online patient portal for results before contacting the laboratory. Please allow up to 48 hours from the time the sample is received by the laboratory for results to be available
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COVID-19 Testing FAQs
Aegis’ COVID-19 testing FAQ provides information specific to our lab’s testing capabilities. Additionally, the FAQ document summarizes pertinent information from various resources leading the fight against the COVID-19 pandemic.
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COVID-19 Fact Sheets
Aegis' COVID-19 Fact Sheets provides information for patients and providers regarding the risks and benefits of using Aegis' tests for COVID-19. For both providers and patients, the test used can be found in the "Sample Comments" section of the Laboratory Report.
Patient Fact Sheet
This Fact Sheet contains information to help you understand the risks and benefits of using this test for the diagnosis of COVID-19. After reading this Fact Sheet, if you have questions or would like to discuss the information provided, please talk to your healthcare provider.
Taq-Path™ COVID-19 Combo Kit : Download Patient Fact Sheet (English - Taq-Path)
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Antibody Patient Fact Sheet
This Fact Sheet contains information pertaining to testing for antibodies to the virus that causes Coronavirus Disease 2019 (COVID-19) using a SARS-CoV-2 Antibody Test that was authorized for emergency use by FDA. After reading this Fact Sheet, if you have questions or would like to discuss the information provided, please talk to your healthcare provider.
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This Fact Sheet informs you of the significant known and potential risks and benefits of the emergency use of a Molecular Laboratory Developed Test (LDT) COVID-19 Authorized Test (Molecular LDT COVID-19 Authorized Test) that has been issued an Emergency Use Authorization (EUA) by FDA. The Molecular LDT COVID-19 Authorized Test is authorized for use on respiratory specimens collected from individuals suspected of COVID-19 by their healthcare provider.
Taq-Path™COVID-19 Combo Kit : Download Provider Fact Sheet (English - Taq-Path)
SARS-CoV-2 qPCR - Aegis Sciences Corporation : Download Provider Fact Sheet (English - Aegis Sciences Corporation)
Antibody Provider Fact Sheet
This Fact Sheet informs you of the significant known and potential risks and benefits of the emergency use of certain SARS-CoV-2 Antibody Tests.