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COVID-19: Antibody testing

 

Serological tests detect evidence of the body’s immune response to an infection, which can provide information on both current and prior infection and on vaccine-induced immune responses. 

Several types of serological tests are available. In the context of SARS-CoV-2, rapid diagnostic tests (RDTs), enzyme-linked immunosorbent assays (ELISAs),  electrochemiluminscence assays (ECLIA) and chemiluminescent microparticle immunoassays (CMIA) have been designed to detect antibodies specific to the spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins of the SARS-CoV-2 virus. 

Rapid diagnostic tests (RDTs) rely on a lateral flow assay format that returns qualitative (positive or negative) results within minutes. A small blood sample is placed at one end of the test strip, and the antibodies of interest in the blood sample interact with tagged proteins embedded in the test. The test displays colored lines at the end of the strip corresponding to a positive, negative, or inconclusive result with respect to the presence of the desired antibodies. RDTs are not capable of providing quantitative results indicating the amount of the antibodies in the specimen. They are small, portable, and can be used at point of-care (POC). In the context of COVID-19, RDTs most frequently test for the presence of patient antibodies (IgM and IgG) specific to SARS-CoV-2.

Enzyme-linked immunosorbent assays (ELISAs) rely on specific binding of patient antibodies to a fixed viral protein of interest, often in a 96-well plate. ELISAs can return qualitative or quantitative results and are generally performed in a lab setting. These tests use whole blood, plasma, or serum samples. Patient samples are incubated with the viral protein of interest to allow antibody-protein binding. The resulting antibody-protein complexes are then exposed to a second antibody or a substrate that produces a color or fluorescent-based signal when bound to the complexes. The resulting signal reflects the presence and/or level of specific antibodies in the patient sample. In the context of COVID-19, ELISAs most frequently test for patient antibodies (IgM and IgG).

Electrochemiluminscence assays (ECLIA) rely on the binding of specific antibodies in a patient’s serum or plasma sample to specific antigens in reaction wells on an automated analyser. The patient’s sample is incubated with specific recombinant antigens, one of which is labelled with a ruthenium complex; this results in antibody/antigen complexes being formed. Streptavidin-coated microparticles are then added to the reaction well which bind the antigen/antibodies complexes to this solid phase.  The reaction mixture is then aspirated into the measuring cell, where the microparticles are magnetically captured onto the surface of the electrode; any unbound substances are then removed.  Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. The resulting signal determines the presence and/or level of specific antibodies in the patient’s sample. These assays can report either qualitatively or quantitatively depending on the assay used. In the context of COVID-19, ECLIA assays most frequently test for Total antibodies (IgG and IgM)

Chemiluminescent Microparticle Immunoassays (CMIA) rely on the binding of specific antibodies in a patient’s serum or plasma to specific antigens in reaction wells on an automated analyser. The patient’s sample is incubated with antigen coated paramagnetic microparticles and assay diluent; this results in the formation of antibody/antigen complexes. Following a wash cycle, a second antibody is then added to the reaction well and incubated. After a second wash cycle, a substrate is added to the reaction well and the resulting chemiluminescent reaction is measured as relative light units by the system optics. The resulting signal determines the presence and/or level of specific antibodies in the patient’s sample. These assay reports either qualitatively. In the context of COVID-19, CMIA assays most frequently test for patient’s antibodies (IgG and IgM).

Genomic surveillance of SARS-COV-2 has shown some genetic variation that may influence the specificity of the antibody testing, as well as having an influence on the vaccine programme. 

Neutralisation assays provide quantitative information on the ability of patient antibodies to confer protective immunity. They are the most time- consuming and skill-based of the serological tests. Using cell culture, live virus, and patient antibodies, researchers can visualise and quantify in a patient sample the level of antibodies capable of blocking viral replication. These tests require whole blood, serum, or plasma samples from the patient. Because these tests require live virus to challenge the antibodies, neutralisation assays must be performed in the appropriate biosafety containment level (Biosafety Level 3, or BSL-3, or above) and require a week or longer to return results.

 

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