COVID-19 IgG Dual Antibody Test FAQ
The following is a list of frequently asked questions relating to ZRT’s COVID-19 IgG Dual Antibody Test.
The US Food and Drug Administration (FDA) has supported the development of diagnostic molecular (PCR) and serologic (antibody) testing for COVID-19 (Coronavirus disease 2019) under an emergency testing policy for the duration of the COVID-19 pandemic. The FDA hopes to accelerate COVID-19 testing while ensuring that tests are performing properly. Antibody testing requires assay manufacturers to go through a rigorous Emergency Use Authorization (EUA) process to distribute testing. No EUA is required currently for laboratory developed tests (LDT) if they are only run at the high-complexity laboratory that developed the test, and the original assays used for development currently have, or are seeking, EUA approval. At this time, only health care provider assisted collections are allowed for LDTs, while at-home collection will require an EUA. The FDA still has not released an EUA template for at-home collection, but ZRT is actively working with the FDA to achieve this type of sample collection when more information is released. The FDA has not approved any molecular or serology testing, and all current authorized tests are only for emergency use during the duration of the COVID-19 pandemic.
A laboratory developed test (LDT) is a diagnostic test that is validated and run at a single high-complexity laboratory. LDTs cannot be run at another laboratory without full validation. This generally involves modifying a manufacturer’s test kit to work with alternative sample types. Almost all tests developed at ZRT are LDTs, and we have over 20 years of experience working with alternate samples types such as dried blood spot, dried urine and saliva.
There are multiple immunoglobulin classes that are used for SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) antibody testing, and each of those can be against a different viral protein. The most common are immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG), and the most common viral proteins to test against are the S1 spike (S1), S2 spike (S2), receptor binding domain (RBD), and the nucleocapsid (N) proteins. When the virus dies and fragments, the body recognizes the different viral proteins and creates antibodies against them. IgA and IgM are believed to be earlier and shorter-lived antibody responses, while IgG may be delayed in comparison, but the response is longer lived and is often associated with long term immunity. Research has shown that IgG antibody assays have higher sensitivity and specificity compared to IgA and IgM. ZRT Laboratory tests for two separate IgG antibodies, one against the surface S1 spike protein and the other against the internal nucleocapsid protein that wraps the viral DNA. The literature has indicated that antibody responses can vary significantly from person to person and responses against one viral protein may be stronger and last longer than another.
Antibodies against SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) can be detected using lateral flow (LFA), enzyme linked (ELISA), or chemiluminescent (CLIA) immunoassays. Lateral flow (cartridge) assays are quick, relatively simple, and can be used at the point-of care, but they are prone to false positives, misinterpreted results, and lack true positive and negative controls to monitor assay performance. ELISA assays are considered to have the highest specificity (less likely to identify antibodies to a similar virus) for SARS-CoV-2 IgG antibodies and allow for controls to be run in parallel with patient samples. ZRT’s COVID-19 IgG Dual Antibody Test uses ELISA technology, which is run at our high-complexity laboratory, and includes positive/negative controls on every run.
ZRT laboratory currently participates in SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) IgG proficiency testing though the College of American Pathologists (CAP).
ZRT’s COVID-19 IgG Dual Antibody Test can be used to confirm or identify prior COVID-19 (Coronavirus disease 2019) cases. If you were unable to get molecular (PCR) testing while showing symptoms of COVID-19, an antibody test should be taken 20+ days from when symptoms first started. Antibody testing should not be used to officially clear people for work at this time. It is unclear if there is long term immunity (>6 months), and whether IgG antibodies and/or cell memory (T-Cells) are protective. If you think you currently have COVID-19, please talk with a healthcare provider who can direct you to molecular (PCR) testing.
It is possible to have COVID-19 (Coronavirus disease 2019) and be asymptomatic. The only ways to know if you were infected is to take a molecular (PCR) test while infected, or test for antibodies 20+ days after symptoms begin. Asymptomatic individuals typically don’t develop high levels of antibodies, but results can vary significantly from person to person.
A more useful test if you believe you were recently exposed to COVID-19 (Coronavirus disease 2019) is a molecular (PCR) test. IgG antibodies will not be present at detectable levels until 1-3 weeks after COVID-19 symptoms show, so an antibody test is not useful at this time.
Testing for COVID-19 (Coronavirus disease 2019) antibodies is a way to tell if you were exposed to SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) sometime in the past. If molecular (PCR) testing was not available to detect a current infection, or you want to determine if you developed or still have a detectable antibody response, antibody testing is useful. IgG antibody levels will rapidly rise (seroconversion), and slowly decline over time. Antibodies to the original SARS-CoV virus lasted from 4 months to 2 years. Early studies have suggested that COVID-19 antibodies may help neutralize or prevent rapid spread of the virus in the case of a second infection. We are still learning if immunity is possible, and how long it will last.
It is highly unlikely that COVID-19 (Coronavirus disease 2019) was circulating at significant levels in the United States prior to February 2020. The 2019/2020 flu season was exceptionally bad, and COVID-19 and the flu show similar symptoms. ZRT Laboratory is finding in our IRB-approved study that a majority of people who thought they had COVID-19 likely did not. This is also evident based on the low number of positive molecular (PCR) tests compared to the number that are run.
Antibody testing should be done 20+ days after the first COVID-19 (Coronavirus disease 2019) symptoms are present. If you are curious if you had COVID-19 and were asymptomatic, then the antibody test can be completed at any time and frequency. It is also possible to re-test to see how a positive antibody response has increased or decreased over time.
If a COVID-19 (Coronavirus disease 2019) infection was confirmed with molecular (PCR) testing, then testing for antibodies 20+ days after symptoms first appear and at least 7 days from the end of symptoms will show if there was an antibody response to the virus. It is unclear at this time if antibodies indicate immunity against SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). However, there may be reasons to see if you have antibodies to COVID including the ability to donate plasma.
Children can be infected by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) and can develop an antibody response like adults. A finger stick antibody test is less invasive than a venous serum draw, making it a great option for testing children.
Dried blood spot testing has been around for over a half century and is a great way to preserve, ship, and store samples. Unlike serum, plasma and whole blood, dried blood spot SARS-CoV-2 IgG antibodies are stable at room temperature for at least 30 days. Dried blood spot testing is used around the world where refrigerated transportation is not readily available and is rapidly catching on as an ideal sample type for blood collection.
Dried blood spot testing is a simple and convenient alternative to a venous serum draw. Most patients report minimal discomfort from a finger stick, and our study results show excellent concordance between matching serum and dried blood spot samples. Dried blood spot samples are easy to ship, and do not require refrigeration. If re-testing is needed, samples are stored for that purpose for at least 30 days before discarding.
Dried blood spot collection is safe for all age groups, and lancets are commonly used on the heels of neonates for dried blood spot collection.
ZRT Laboratory examined multiple transportation, storage, and stability conditions during our validation. Dried blood spots are stable at room temperature and many other lower and higher temperature conditions for at least 1 month. A dried blood spot sample, when properly dried and packaged in a Ziploc plastic bag with the included desiccant, will be stable during shipping and have no effect on results.
Current research shows that there is likely no live SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) virus present in blood. Oral fluids and feces have both been shown to carry live virus. Viral RNA has been detected in a small number of blood samples, but this is believed to be non-transmittable remnants of fragmented virus.
Molecular (PCR) testing looks for SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) RNA (live or dead virus) in respiratory specimens (nasal/oral) and can be detected before COVID-19 (Coronavirus disease 2019) symptoms are present. A positive PCR test indicates a current or very recent infection. Antibody testing using serum, plasma, whole blood or dried blood spot (DBS) looks at the antibodies your body produces against SARS-CoV-2, which only begins after symptoms show. Most people will develop antibodies (seroconvert) within 1-2 weeks of symptoms, but others can take 3-4 weeks. Essential PCR testing is looking for current or very recent infection while antibody testing is looking for past infections and potential immunity. If you are currently experiencing COVID-19 symptoms, then PCR testing is important. If you have recovered from COVID-19 or possible COVID-19 then an antibody test 20+ days from when symptoms started is useful. Information compiled from the SARS virus, which shows some similarities to SARS-CoV-2, showed IgG antibodies lasting up to two years.
Studies have shown that every person has a different antibody response when infected by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). It is possible that a strong antibody response might mount against viral nucleocapsid protein but not the S1 spike protein. The opposite is true as well. Research has also shown that antibodies against the viral nucleocapsid protein may be present before the S1 spike protein. The added benefit of testing antibodies against both S1 spike and nucleocapsid antibodies is that a second test is being run without a reflex test, which is only useful if the first test is positive. ZRT Laboratory hopes to reduce the number of false positives and negatives and increase testing confidence by looking at two separate IgG antibodies.
There are multiple immunoglobulin classes that are used for SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) antibody testing, and each of those can be against a different viral protein. Immunoglobulin A, M and G (IgA, IgM, and IgG) are the most common antibodies and the S1 spike (S1), S2 spike (S2), receptor binding domain (RBD), and the nucleocapsid (N) proteins are the viral proteins commonly tested against. When the virus dies and fragments, the body recognizes different viral proteins and creates antibodies against them. IgA and IgM antibodies are believed to be earlier and shorter-lived, while IgG antibodies may be delayed in comparison, but the response is longer lived and is often associated with long term immunity. Research has shown that IgG antibody assays have higher sensitivity and specificity compared to IgA and IgM. ZRT Laboratory’s assay looks at the IgG antibody response against both the viral S1 spike and nucleocapsid proteins. The literature has indicated that antibody responses can vary significantly from person to person and responses against one viral protein may be stronger than another.
ZRT’s COVID-19 IgG Dual Antibody results will indicate a past COVID-19 (Coronavirus disease 2019) infection. This is useful to determine possible exposure if molecular (RT-PCR) testing was not available before or during the COVID-19 symptomatic period, or there is some suspicion that the molecular test was flawed (false negative). ZRT’s COVID-19 IgG Dual Antibody test only detects IgG antibodies developed when infected with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), not the earlier and less specific IgA or IgM antibody responses. The IgG antibody response (seroconversion) typically starts within 1-2 weeks of when symptoms first show and may not be detected for 20+ days after symptoms begin. ZRT recommends testing for IgG antibodies 20+ days from symptom onset, and at least 7 days from the end of symptoms. There are reports that some asymptomatic individuals may not develop a detectable antibody response.
Research has shown that antibody responses can vary significantly from person to person. Higher levels of antibodies have been correlated to older age, greater symptom severity, longer symptom duration and increased viral load. In some asymptomatic or very mild cases antibody response may develop and diminish rapidly. Most antibody tests are validated using hospital samples, which are likely to have higher antibody levels due to the severity of cases. ZRT’s ongoing study is looking at a random mix of samples from patients of all age ranges and symptom severity, which we believe is a better indicator of real-life test performance.
Yes, it is possible for a person that is frequently around others to be the only person to test positive for antibodies in the group. It is also possible for nearly everyone at a gathering to catch COVID-19 (Coronavirus disease 2019) from a single person. There is still a lot that is being learned about the spread of COVID-19, and research has shown that viral load plays a big part in that. Some infected with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) are considered super spreaders due to their extremely high viral load, which can be >1000x higher in one person than another. If a person has a low viral load, there is a lower likelihood of viral spread between family members and the public. The problem is, it is near impossible to determine who will be a super spreader, so universal precautions need to be taken such as physical distancing and wearing a mask.
It does not matter what time your sample is collected for the ZRT COVID-19 IgG Dual Antibody Test.
Research has shown that nearly everyone with COVID-19 (Coronavirus disease 2019) developed antibodies by day 20 after symptoms. If you test too early, it is possible that the IgG antibody response (seroconversion) may not have occurred yet.
Most people will recover from a COVID-19 (Coronavirus disease 2019) infection within 14 days of when symptoms first started. For some people, especially those hospitalized, COVID-19 infection can last for months. Testing at least 7 days from the end of symptoms increases the chances of detecting IgG antibodies.
A health care provider assisted collection is up to interpretation at this point. The FDA has not released the definition of a “health care provider” and has recently stated that a live telehealth viewing of the patient collecting the sample is not a substitute for a “health care provider assisted home collection”. The FDA has clarified that a health care provider must assist with the collection in person, either by watching the patient collect or physically assist with the collection. This will be updated as we learn more information from the FDA.
At this time the ZRT COVID-19 IgG Dual Antibody Test cannot be collected at-home without health care provider assistance. The FDA is requiring antibody testing that uses at-home collection to seek emergency use authorization (EUA), for which the FDA doesn’t currently have a template. ZRT Laboratory is currently working with the FDA to determine the best route toward an at-home collection EUA.
ZRT’s COVID-19 IgG Dual Antibody Test has specific test instructions and a digital requisition form that differs from other dried blood spot kits ZRT offers. The instructions include statements required by the FDA. At this time, only samples collected from COVID-19 testing kits will be accepted for antibody testing.
The complete dried blood spot collection should be dried for 4 hours, packaged in the provided Ziploc bag with desiccant, and shipped to the laboratory as soon as possible. A shipment delay should not be a problem, and we recommend that the sample be stored at room temperature out of sunlight until ready to ship.
ZRT’s blood spot collection video can be found here.
ZRT’s turn-around time is usually 3-5 business days.
Yes, it is possible, but rare, to have two non-matching results. There are 8 possible result combinations. Results that don’t match can indicate several different things. One positive and one negative result can indicate a false positive result from a past viral infection, while one indeterminate and one positive can indicate that one antibody response is stronger than the other.
Positive means that antibodies against SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) were detected. Indeterminate means that it is unclear if antibodies against SARS-CoV-2 are present and can indicate a weak response or a past non-COVID-19 (Coronavirus disease 2019) viral infection. Negative means that antibody levels were below the limit of detection.
A false negative result is when a patient is/was infected by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) but their antibody result is negative, while a false positive result is when a patient has not been infected by SARS-CoV-2 but tests positive for antibodies. False positives are likely caused by past viral infections, while false negatives are due to when the test was taken or if an individual has a weak antibody response. There are no reference specimens to work with currently, so the only way to determine a false positive is to test samples collected in 2019 and earlier (prior to first COVID-19 cases) and to cross compare testing. The gold standard now is to compare antibody results with PCR testing, which is flawed because of the possibility of PCR false negatives (false positives with PCR testing only come from reagent or sample contamination). Sensitivity and specificity testing of our IRB-approved study samples indicate a very low number of potential false positives and false negatives. Running two separate antibody tests as we do for ZRT’s COVID-19 Dual Antibody Test helps confirm true positive/negative antibody results.
Currently is it not clear whether detection of IgG antibodies infers immunity to future COVID-19 (Coronavirus disease 2019) infections. Studies have indicated neutralizing ability of IgG antibodies against SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) in animals, but results need to be looked at with caution.
ZRT Laboratory is required to report antibody results to state officials. At this time antibody testing is not being used in case counts for COVID-19 (Coronavirus disease 2019), and there is no use for antibody testing in contact tracing to prevent viral spread.
It is possible have a prior COVID-19 (Coronavirus disease 2019) infection and to test negative for antibodies. Technically this is considered a false negative, but it is a valid result and not caused by a problem with the performance of the antibody test. It is unclear how long an IgG antibody response lasts, but it is likely that a weak antibody response will become undetectable within months of infection. A strong antibody response is likely to last for months to years.
Results are provided through ZRT’s patient/provider portal.
Sensitivity and specificity are used to describe the performance of a serology assay. Sensitivity is the assay’s ability to correctly identify patients with antibodies (true positive rate). Specificity is the assay’s ability to correctly identify patients without antibodies (true negative rate). PCR molecular testing is used to confirm a true positive/negative result, but it is possible for there to be false negatives with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) PCR testing, so results must be looked at with caution.
See our Provider Data Sheet and the tables below for more information
The positive and negative predictive value is used to determine the proportion of true positive and true negative results. These numbers are determined based on sensitivity and specificity results, and the positive/negative predictive value was 95.2%/100% for the ZRT dried blood spot (DBS) SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) IgG S1 Spike assay and 95.2%/93.8% for the ZRT DBS SARS-CoV-2 IgG Nucleocapsid assay. PCR molecular testing is used to confirm a true positive/negative result, but it is possible for there to be false negatives with SARS-CoV-2 PCR testing, so results must be looked at with caution.
Positive/negative concordance is used to compare two assay methods. In this case, the ZRT dried blood spot (DBS) SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) IgG S1 Spike assay was compared to the EUROIMMUN Serum SARS-CoV-2 IgG S1 Spike assay, and the ZRT DBS SARS-CoV-2 IgG Nucleocapsid assay was compared to the Epitope Diagnostics (EDI) Serum SARS-CoV-2 IgG Nucleocapsid assay. The serum assays were run according to manufacturer recommendations.
See our Provider Data Sheet and the tables below for more information
ZRTs validation for our dried blood spot COVID-19 IgG Dual Antibody Test involved collection and analysis of matching serum and dried blood spot samples collected under our Institutional Review Board (IRB) approved study. Our complete validation included within-assay precision, inter-assay precision, sample stability under different conditions, blood spot size, punch location, hematocrit effects, interferences, hook effect, drying and shipping conditions, filter paper lot analysis, blood spot dry time, IgG lateral flow vs ELISA comparison, serum vs dried blood spot absorbance comparison, sensitivity/specificity, positive/negative concordance, and establishment of calibrators and controls. ZRT’s laboratory-developed test (LDT) validations are extensive, and go well beyond what is typically required for a LDT. We plan to publish this information after the completion of our study.