An introduction to antibody testing for COVID-19
Antibodies are specific proteins created as an immune response against infections which activate the complement system to destroy bacterial cells by lysis. And antibodies facilitate phagocytosis of foreign substances by phagocytic cells. They can be detected in the blood of people who were infected with SARS-CoV-2.
Figure 1. Antibodies are proteins created as part of an immune response to SARS-CoV-2 infection. They can be detected in the blood of people infected with SARS-CoV-2 regardless of whether they were symptomatic or asymptomatic.
An antibody test helps determine if an individual was ever infected with SARS-CoV-2, regardless of whether they were symptomatic or asymptomatic. It’s important to note that antibody tests are only able to detect past SARS-CoV-2 virus infections indirectly, by measuring the host immune response to the virus. Another test, namely RT-PCR is the primary tool for diagnosing active SARS-CoV-2 infections.
What are SARS-CoV-2 antibody tests?
There are three components of an antibody test:
- The antibody class detected—either immunoglobulin G (IgG) antibodies alone, or in the case of total antibody tests, IgG antibodies with immunoglobulin M (IgM) antibodies.
- The SARS-CoV-2 antigens used to design the assay—purified proteins of SARS-CoV-2, not the live virus.
- The patient samples—collected by venipuncture and may include whole blood, plasma or serum.
Because these tests do not use the live virus, they can be performed in lower biosafety level laboratories (BSL-2).
Figure 2. The three components of an antibody test are the antibody class detected, the SARS-CoV-2 antigens used to design the assay, and the patient specimen.
Tests that detect antibodies to SARS-CoV-2 fall into two categories:
- Enzyme-linked immunosorbent assay (ELISA)
- Point-of-care lateral flow immunoassays
In an enzyme-linked immunosorbent assay, or ELISA, viral proteins, or antigens, are immobilized on a microwell plastic plate and the patient samples are added.
Figure 3. Enzyme-linked immunosorbent assay (ELISA) combines SARS-CoV-2 antigens and a patient sample on a microwell plastic plate.
The samples are incubated with the viral protein-coated plate, and if the patient has antibodies to the viral protein, the antibody and antigen bind together. IgG antibodies that target human antibodies then bind to the antibody-protein complex. These anti-human antibodies have an enzyme attached to them, so that when a substrate is added, it causes a change in colour. The bound antibody-protein complex can be detected by measuring the change in color with an automated plate reader.
Figure 4. In this enzyme-linked immunosorbent assay, or ELISA, microplate, the patient blood sample has been combined with SARS-CoV-2 antigens. SARS-CoV-2 antibodies in the sample bind to the antigens. Then IgG antibodies with an attached enzyme bind to the antigen-antibody complex and change color when a substrate is added. The antigen-antibody complex can be detected by measuring color change.
If the COVID-specific antibodies are not present in the patient sample, the anti-human antibodies don’t bind to the plate and are washed away, so there is no colour change when the substrate is added.
ELISAs require trained laboratory technicians and specialized instruments. These immunoassays can be automated, with many patient samples processed at the same time, but the time required varies by test method. All currently authorized COVID-19 antibody tests are qualitative and provide a result that is positive, negative, or indeterminate, rather than quantitatively measuring the actual antibody level.
Figure 5. Trained laboratory technicians and specialized instruments are required to perform enzyme-linked immunosorbent assays (ELISA).
Lateral flow immunoassays
Lateral flow immunoassays (LFIA) are also known as rapid serologic tests, or rapid diagnostic tests (RDTs). Samples are placed onto test strips to detect SARS-CoV-2 antibodies. Let’s consider the architecture of the test strip more closely.
There are several important regions on the test strip. The conjugate pad contains antigens labelled with gold and gold-tagged control antibodies—usually IgG antibodies from an animal source. The gold labelling will make the bound antibodies and target antibodies visible on the control and test lines. The test line contains antibodies to detect the SARS-CoV-2 antibodies of interest in the patient sample. The control line contains pre-attached antibodies to bind the gold-tagged control antibodies and show the test is working properly.
The patient sample is spotted on the paper-like membrane and the serum contents are drawn across by capillary effect (Fig. 6a). If the target antibody is present in the patient sample, the gold-labelled antigen in the conjugate pad will bind to the target antibodies (Fig. 6b) and they will migrate along the test strip together until the target antibodies bind to the pre-attached antibodies on the test line (Fig. 6c). The gold-labelled control antibodies will migrate further along to bind to the pre-attached antibodies on the control line (Fig. 6d).
Figure 6. Lateral flow immmunoassays (LFIA), or rapid diagnostic tests (RDTs), can be used to detect SARS-CoV-2 antibodies in a blood sample, saliva sample or nasal swab: a) the sample is spotted on the paper-like membrane and the serum contents are drawn across by capillary effect, b) gold-labelled antigens in the conjugate pad bind to target antibodies in the sample and continue to migrate along the test strip, c) target antibodies also bind to the pre-attached antibodies on the test line, d) gold-labelled control antibodies migrate from the conjugate pad and bind to the pre-attached antibodies on the control line.
The test then shows colored lines, similar to a home pregnancy test, to indicate positive or negative results for antibodies to SARS-CoV-2. A positive result will show a colored test line and control line while a negative result will show only a colored control line. Inconclusive results will show no lines or possibly a coloured test line without a functioning control line.
Figure 7. A positive result on a lateral flow immunoassay (LFIA) will show two lines—a test line and a control line—while a negative test will show only a control line and inconclusive results will show no lines or a test line without a control line.
LFIAs are single-use and rely on visual interpretation. They can be performed on blood samples obtained by fingerstick rather than venipuncture. Other possible patient samples include saliva or nasal swab. The time required for testing can be as short as 15–30 minutes.
These tests have the potential for point of care usage, or home self-testing, without requiring a laboratory, but they are not approved for personal use yet.
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