Background

Serology testing or Antibody testing for SARS-CoV-2 is at increased demand in order to better quantify the number of cases of COVID-19, including those that may be asymptomatic or have recovered. Serology tests are blood-based tests that can be used to identify whether people have been exposed to a particular pathogen by looking at their immune response. In contrast, the RT-PCR tests currently being used globally to diagnose cases of COVID-19 can only indicate the presence of viral material during infection and will not indicate if a person was infected and subsequently recovered. These tests can give greater detail into the prevalence of a disease in a population by identifying individuals who have developed antibodies to the virus.

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.

Development of Antibodies and Immunity

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.In addition, development of neutralizing antibodies can also be assessed. Neutralizing antibodies inhibit viral replication in vitro, and as with many infectious diseases, their presence correlates with immunity to future infection, at least temporarily.

Recurrence of COVID-19 illness appears to be very uncommon, suggesting that the presence of antibodies could confer at least short-term immunity to infection with SARS-CoV-2. Consistent with this observation, experimental primary infection in primates and subsequent development of antibodies resulted in protection from reinfection after the primates were rechallenged. Additionally, antibody development in humans correlates with a marked decrease in viral load in the respiratory tract. Taken together, these observations suggest that the presence of antibodies may decrease a person’s infectiousness and offer some level of protection from reinfection. However, definitive data are lacking, and it remains uncertain whether individuals with antibodies (neutralizing or total) are protected against reinfection with SARS-CoV-2, and if so, what concentration of antibodies is needed to confer protection.

Types of Antibody Testing

Different types of assays 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.

Rapid diagnostic test (RDT):

This is typically a qualitative (positive or negative) lateral flow assay that is small, portable, and can be used at point of care (POC). These tests may use blood samples from a finger prick, saliva samples, or nasal swab fluids. RDTs are often similar to pregnancy tests, in that the test shows the user colored lines to indicate positive or negative results. In the context of COVID-19, these tests most frequently test for patient antibodies (IgG and IgM), or viral antigen. In some cases, it can be beneficial to measure baseline (before infection) of IgG and IgM titers.

Chemiluminescent immunoassay:

This test is typically quantitative, lab-based, and uses whole blood, plasma, or serum samples from patients. A variation of this test can use magnetic, protein-coated microparticles, known as a chemiluminescent microparticle immunoassay. The test relies on mixing patient samples with a known viral protein, buffer reagents, and specific enzyme-labeled antibodies that allow a light-based, luminescent read-out. Any antibodies in the patient sample that react to the viral protein will form a complex. Then, (secondary) enzyme-labeled antibodies are added that bind to these complexes.

Neutralization assay:

This test relies on patient antibodies to prevent viral infection of cells in a lab setting. Neutralization assays can tell researchers if a patient has antibodies that are active and effective against the virus, even if they have already cleared the infection. These tests require whole blood, serum, or plasma samples from the patient. Neutralization assays depend on cell culture, a lab-based method of culturing cells that allow SARS-CoV-2 growth (like VeroE6 cells). When virus and cells are grown with decreasing concentrations of patient antibodies, researchers can visualize and quantify how many antibodies in the patient serum are able to block virus replication. This blocking action can happen through the antibody binding to an important cell entry protein on the virus, for example.

Enzyme-linked immunosorbent assay (ELISA):

This test can be qualitative or quantitative and is generally a lab-based test. These tests usually use whole blood, plasma, or serum samples from patients. The test relies on a plate that is coated with a viral protein of interest, such as Spike protein. Patient samples are then incubated with the protein, and if the patient has antibodies to the viral protein they bind together. The bound antibody-protein complex can then be detected with another wash of antibodies that produce a color or fluorescent-based readout. In the context of COVID-19, these tests most frequently test for patient antibodies (IgG and IgM).

How Does an Antibody Test Work?

You’ll have to give blood, like through a finger prick. Doctors test for two kinds of antibodies to SARS-CoV-2. They look for:
  • IgM antibodies, which develop early in an infection, and
  • IgG antibodies, which are more likely to show up later after you’ve recovered

It takes your body about 4 weeks to develop IgM antibodies. But scientists aren’t sure how long it’ll take for this to happen with SARS-CoV-2. More tests are needed to find out.

Keep in mind that current antibody tests can’t tell you if you’re immune to COVID-19. That’s because we don’t know how long these antibodies might protect you against the coronavirus. And these tests shouldn’t be used to diagnose the virus.

A technician will take a bit of your blood, like through a finger prick. The test looks for one or both kinds of antibodies to SARS-CoV-2, the virus that causes COVID-19 as mentioned above.Most people have IgG antibodies about 7 to 10 days after COVID-19 symptoms start. They usually stay in your blood long after the infection goes away, but we don’t know how long that is for the new coronavirus.

Antibody Test for IgG

This test detects IgG antibodies that develop in most patients within seven to 10 days after COVID-19 symptoms begin. IgG antibodies remain in the blood after an infection has passed. These antibodies indicate that you may have had COVID-19 in the recent past and have developed antibodies that may protect you from future infection.It is unknown at this point how much protection antibodies might provide against reinfection.

Antibody Test for IgM

This test detects IgM antibodies. IgM is usually the first antibody produced by the immune system when a virus attacks. A positive IgM test indicates that you may have been infected and that your immune system has started responding to the virus.  When IgM is detected you may still be infected, or you may have recently recovered from a COVID-19 infection.

Testing and Results

The utility of tests depends on the sensitivity and specificity of the assays; these performance characteristics are determined by using a defined set of negative and positive samples. In addition, the predictive values of a test should be considered because these values affect the overall outcome of testing. Positive predictive value is the probability that individuals with positive test results are truly antibody positive. Negative predictive value is the probability that individuals with negative test results are truly antibody negative. Positive and negative predictive values are determined by the percentage of truly antibody positive individuals in the tested population (prevalence, pre-test probability) and the sensitivity and specificity of the test. For example:In a high-prevalence setting, the positive predictive value increases — meaning it is more likely that persons who test positive are truly antibody positive – than if the test is performed in a population with low-prevalence. When a test is used in a population where prevalence is low, the positive predictive value drops because there are more false-positive results, since the pre-test probability is low. Likewise, negative predictive value is also affected by prevalence. In a high-prevalence setting, the negative predictive value declines whereas in a low-prevalence setting, it increases.

What’s the Difference Between a Coronavirus Test and an Antibody Test?

A coronavirus test, sometimes called a diagnostic test, looks for signs of active virus. It’s simpler and faster than an antibody test. But it tells you only if you have the virus in your body at the moment when you’re tested.An antibody test shows that you had the virus at some point in the past. It could be gone, or you could still be contagious.

Interpreting Results When Symptomatic at the Time of Testing

Both IgM and IgG are Negative

The subject’s immune system has not produced any antibodies to the target viral antigen. If it has been greater than 7 days since the onset of fever or other symptoms, the disease is unlikely to be COVID-19, but a full-panel test including COVID-19, influenza, and bacterial bronchitis could be performed if available and recommended based on history and symptoms. If it has been less than 7 days since the onset of symptoms, COVID-19 cannot be ruled out with antibody testing alone. The subject should isolate from others and could repeat antibody testing at least 7 days after the onset of symptoms unless a molecular test was available for confirmation. If symptoms are severe and COVID-19 is suspected based on clinical signs, a molecular test is indicated for the detection of viral genetic material in respiratory samples. The subject should not return to normal activities until SARS-CoV-2 infection is ruled out through alternate diagnoses, molecular testing or repeat antibody testing, or appropriate time has elapsed after symptom development to rule out ongoing viral shedding (shedding can occur up to approximately 31 days from exposure).

IgG is Positive and IgM is Negative

The subject’s immune system has produced antibodies to the target viral antigen. The subject is likely in the later stages of the disease course but may still be contagious to others and capable of spreading the virus. The subject should remain isolated from disease-negative population for at least 14 days to minimize the chance of spreading the virus. If more rapid return to community activities is warranted, for example, for critical industry workers, a molecular test should be performed to assess viral shedding status.

IgM is Positive and IgG is Negative

The subject’s immune system is actively producing antibodies to a recent infection with the target virus. The subject should immediately isolate from healthy individuals and seek medical care appropriate to the symptom severity they experience. The subject can spread the disease to others at this point in disease course. After at least 14 to 21 days, the subject should consider repeat testing to determine IgG status before returning to normal activities.

Both IgM and IgG are Positive

The subject’s immune system is actively producing antibodies to an ongoing infection that likely began more than 14 days ago. The subject should immediately isolate from healthy individuals and seek further medical care appropriate to the symptom severity they experience. The subject can likely still spread the disease to others. Consider repeat testing in 7 to 14 days to confirm IgG only status before returning to normal activities. If more rapid return to community activities is warranted, for example, for critical industry workers, a molecular test should be performed to assess viral shedding status.

Case Studies

Read the medical study of a COVID-19 IgM and IgG rapid test; an effective tool for assessing past exposure to SARS-CoV-2, a Journal article conducted by Tove Hoffman, Karolina Nissen, Janina Krambrich, Bengt Rönnberg, Dario Akaberi, Mouna Esmaeilzadeh, Erik Salaneck, Johanna Lindahl & Åke Lundkvist (2020).

Read the medical study “Understanding Antibody Testing for COVID-19” ,a Medical Journal article conducted by David Jacofsky(MD,a), Emilia M. Jacofsky(PhD,b) and Marc Jacofsky (PhDc) (2020).