Introduction to Chemiluminescence immunoassays
Serological tests are used to gain a deeper understanding of the immune response to pathogens and the tests help maintain community health by checking for antibodies in human biological samples. Chemiluminescence is a widely used system of reporting binding events. It is preferred because it uses a simple device for measurement, often one that measures output of visible light. This also allows the process to have a wide dynamic range, detecting light from binding events whether the sample is dilute or concentrated. Such detection is done with high sensitivity and with low background noise. The chemiluminescent magnetic microparticle immunoassay (CMIA) is a method developed to bring together the advantages of chemiluminescence and magnetic particles for immunoassays.
General protocol for Chemiluminescence serological tests
The chemiluminescence serological test begins with either a primary antibody bound to the surface of microplate, likely made of polystyrene, or magnetic microparticles (beads) in solution. The microparticles are pre-conjugated with primary antibodies. The primary antibodies bind to antigen in a sample. Then a secondary antibody with a chemiluminescent enzyme bound to it, such as HRP, is bound to the antigen. When substrate is added to the sample, the enzyme catalyses a reaction that emits light that can be seen and measured. There are intermediate wash steps for this procedure which can be done efficiently when using magnetic beads.
Chemiluminescent Serological tests for SARS-CoV-2 detection
Companies have been exploring and innovating chemiluminescent serological tests for use in detection of SARS-CoV-2 during the current pandemic. Most researchers and companies are interested in the adaptive immune response for diagnostics and are trying to detect two isotypes of immunoglobulins, IgM and IgG. IgM is present days after infection while IgG is present weeks after infection. These timelines and concentrations are used to understand at which stage of infection a person is in when they are tested for disease after exhibiting symptoms. An example of such a test is a chemiluminescent microparticle immunoassay offered by Abbott, which uses CMIA’s for qualitative detection of the immunoglobulin isotype IgG in patient samples of serum or blood.
Recent advancement have demonstrated that such tests can also demonstrate immunity to variants that emerge through mutations.
Example of a commercial Chemiluminescent serological test
Serological assays for SARS-CoV-2 are being developed and sold by many companies, one example is the biotech company Roche Diagonstics. They have developed an electrochemiluminescent magnetic microparticle immunoassay that has recently been approved by the FDA. For SARS-CoV-2 serological test development, the spike surface protein or nucleocapsid protein are popular targets. For the Roche diagnostics assay, they use nucleocapsid protein as their antigen, the molecule that will bind antibodies from a patient sample. That antigen is pre-bound to two different molecules which produce light when they are in close proximity and substrate is added. The serum of a patient sample is mixed with the pre-bound nucleocapsid, at which point any antibodies in the sample that were produced in response to SARS-CoV-2 will bind to antigen. When substrate is added to the reaction it produces light which can be measured to detect SARS-CoV-2 specific antigens. This assay is unique in that it is optimized to be high specific, and measures high affinity antibodies in serum.
Serological assays are being developed at a rapid rate during the pandemic. Researchers and companies are considering many factors which need to be optimized, such as antigen target, antibody target, detection techniques, and all the steps that lead to successful assays such as purifications and wash steps. Accuracy is an incredibly important factor when considering the large numbers of samples to be tested. Chemiluminescent serological assays using magnetic particles can be done quickly and simply, making them an attractive option when considering how important it is to have accurate tests.
Recent uses with Chemiluminescent tests have revealed how new variants may escape previously acquired immunity. One such study, performed at the São João Hospital, noticed binding to the alpha variants in 74% of samples that originally tested positive for IgG, and only 47% on binding towards the beta variants. Future research may expand on such capabilities to possibly detect which variant an individual may have contracted. Such tools can also be used to measure the effectiveness of vaccine immunity one receives from an injection, with binding tools specifically to vaccine antibodies. This will be a useful tool to observe how protected someone is against new mutations in SARS-CoV-2 as they emerge.
- Protein purification: the step-by-step process
- Magnetic beads vs Agarose beads
- A general filtration process