In the fight against COVID-19, testing of patient samples has been mostly conducted using standard techniques, which has kept clinics struggling to keep up with the demand for testing. The first step in coronavirus testing that needs to be more efficient is the RNA extraction.
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.
In a pandemic it becomes crucial to quickly design and manufacture a diagnostic device for large scale testing of human blood for viruses. Ideally, each step of the diagnosis protocol needs to scalable so that it can be done quickly. The first step, purification, needs to produce pure and clean samples for lower rates of false results. The use of RNA purification kits in coronavirus testing offers a solution to this problem.
There has been a lot of discussion surrounding RNA purification for the purposes for testing people for the presence of viruses from liquid biopsies. Using magnetic beads for the purification, many kits for individual sample preparations are required. At this time there is also potential for use of magnetic beads for large-scale purification of RNA in research towards the development of vaccines and tests.
Chemiluminescence and fluorescence seem like they are the same thing, especially when using them as tracking strategies for magnetic separation in biosensors or in-vitro diagnostic assays. But, they are not the same. Yes, they both give off a photon as an electron relaxes from a higher energy state to a lower energy state, but the difference lies in the method used to excite that electron to a higher energy state in the first place. In fluorescence the electron is kicked up to a higher energy state by the addition of a photon. In chemiluminescence the electron is in a high-energy state due to the creation of anunstable intermediate in a chemical reaction. Light is released when the intermediate breaks down into the final products of the reaction.
The principles of immunoassays
An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. Antibodies made by the adaptive immune response in the body are highly specific towards particular antigens. For example, this is why we receive vaccinations, to help our immune system build an antibody repertoire response towards parts of an antigen before we encounter it in a more pathogenic state. The immunoassay will use those highly specific antibodies to probe for molecules of interest when they are in mixtures with other molecules.
Magnetic beads are used for biomagnetic separation procedures to enrich populations of a target cell, protein, or nucleic acid. Since the affinity between antibody and antigen is strong and specific, antibodies are often conjugated to the surface of magnetic beads in order to bind target cell or protein for enrichment. The magnetic beads are made of polymer (typically polystyrene) and iron oxide particles (usually magnetite (Fe3O4)), and are commercially available in a variety of sizes and surface chemistries. The size of the magnetic bead is important; larger micrometer-sized beads have narrower size distributions and behave more predictably in a magnetic field gradient than smaller nanometer-sized beads. Additionally, the microbeads are better at forming cooperative chains during the magnetic separation process, which improves the efficiency of the separation. When designing or troubleshooting a biomagnetic separation process it is important to evaluate the type of separation rack used and the size of the magnetic beads in addition to the surface chemistry and conjugation procedure. Sometimes the conjugation method is blamed for poor target recovery, but often the problem is due to a poorly designed separation rack.
Column chromatography is a method used in chemistry to isolate a single compound from a mixture. The basic principle of column chromatography is the adsorption of target to the column by designing a column with specific affinity to the target. The target compound adsorbs to the column resin while the remaining mixture easily flow through the column and out the other end. A similar method is used to purify protein and nucleic acids, and it is generally referred to as affinity chromatography. Affinity chromatography requires a solid support (typically a magnetic bead or a resin column) on which to covalently attach a capture molecule which has affinity to the target protein or nucleic acid.
Glutothione S-transferase is a 26 kDa protein that is used as an affinity tag for protein isolation in pull-down assays. The GST tag has specific affinity for the protein glutathione. This means that glutathione can be attached to columns or magnetic beads and used to isolate any protein that has been modified with the gst tag sequence. The modification of proteins with the gst tag sequence is performed in host organisms and results in fusion proteins that consist of the target protein joined by a linker to the 220 amino acids that compose the gst tag.
Ensuring that pharmaceutical products reach the consumer without degradation during shipping and storage has led to the creation of stability testing guidelines. All pharmaceutical products must undergo rigorous and standardized stability tests before they are approved for sale around the world. This has not always been the case for components of In-Vitro Diagnostic (IVD) kits used in clinical and research laboratories worldwide.