Evaluation of COVID-19 IgM and IgG rapid antibody tests

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 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 cannot be used 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

Antibodies are part of our bodies immune response and are, for most of us, produced when the presence of a virus is recognized. This process occurs for all viruses that we are exposed to, including SARS-CoV-2. The antibodies consist of different types including the IgM and IgG antibodies. These are frequently used in different assays as we know more about their response to a virus than we do with for example the IgA antibody.

The IgM antibody is the first antibody to be produced once the immune system recognizes an unwelcomed virus. Research shows that this antibody is detectable within a few days but peaks 14 days after the first outbreak of symptoms. The later developed IgG antibody takes longer to produce but also stays longer in the blood. To have the best chance of detecting IgG antibodies, research suggests that it peaks after 3 weeks since the first symptom and that a test should therefore be taken first then. For SARS-CoV-2 the IgG and IgM antibody does however seem to almost simultaneously be produced. Most experts therefore suggest that an antibody test for SARS-CoV-2 could be taken after 14 days.

How long after the illness onset antibodies remains detectable is not yet known for Covid-19.

Recent studies also show that an antibody tests efficiency can depend on which protein from the coronavirus’s structure the test binds to. The SARS-CoV-2 have different such proteins in its structure, among them the N- and S-protein. Studies reveal that the half-life of an N-protein is notable shorter than of the S-protein. This implies that a test that binds to the S-protein can be more useful if the test is meant to be used over a longer period.

Recurrence of COVID-19 illness appears to be very uncommon, even if exceptional cases have been reported. The common theory is however that the presence of antibodies confers 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 again exposed to the infection. 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 develops 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

Antibody tests is a group of tests consisting of different type of assays. On a general note, one can say that an antibody test can be classified to detect either binding or neutralizing antibodies. We will explain more about the different types of tests below:

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 the sense that the test shows the user coloured 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 titres.

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-labelled 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, enzyme-labelled 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, for example, 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 lab-based. 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 the 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 colour or fluorescent-based readout. In the context of COVID-19, these tests most frequently test for patient antibodies (IgG and IgM).

Testing and Results

Two central measures that decide the quality of a Covid-19 test is the sensitivity and specificity. These performance indicators are determined by using a defined set of negative and positive samples. A test with high specificity means that the risk of receiving a false positive test result is low. In regards of antibody testing, the specificity is extremely important as a positive test result implies that the person suffers lower risk of being reinfected. A falsely positive result: saying that the patient has antibodies when he or she in fact does not is more troubling than the opposite – receiving a negative test result when in fact the patient has developed antibodies. The sensitivity of a test is still very important, as providing patients with truthful test results is the overall target with testing, but a false negative test result has less severe implications for the patient.

In addition, the predictive values of a test should be considered since 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  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.