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.
Magnetic microbeads are used to enrich cells, proteins, and nucleic acids from complex samples using biomagnetic separation. The process requires a well-designed magnetic separation rack, and the beads need to be coated and functionalized in order to capture the desired target molecule. Magnetic microbeads are typically made of iron oxide (Fe3O4) also known as magnetite, and are 0.5 to 500 μm in diameter. The diameter of the microbeads is a function of the non-ferrous material composing the beads.(more information below). The microbeads are small enough that they are superparamagnetic, which means that they are inherently non-magnetic, but they become magnetized when placed into a magnetic field. This effect is reversible, and the magnetism disappears again after the microbeads are removed from the magnetic field.
A sonicator bath is a tool that propagates ultrasonic waves through fluid contained within it. The ultrasonic bath is used in the laboratory to lyse cells, to degass water, and to break up clumped and aggregated magnetic beads, among many other uses. Ultrasonic cleaners are used to remove dirt and grime on objects that are hidden in difficult crevices that brushes or sprays cannot access. The most common fluid used in an ultrasonic bath or ultrasonic cleaner is distilled water. Other solvents may be added to help in cleaning processes, but in the laboratory, sonicator baths are almost always filled with distilled water. One must be careful when using solvents to ensure that they don’t have a low flash point as the ultrasonic waves will heat up the fluid in the bath. Sonicator baths work by applying ultrasonic waves to fluid. Ultrasonic waves are sound waves greater than 20 kHz; when propagated through fluid they bounce into air bubbles and cause them to burst. The shock wave released by bursting air bubbles helps to lyse cells, remove dirt from surfaces, or to break apart aggregated magnetic beads.
Recrystallization is the process of obtaining pure crystals of a compound from a solution containing impurities. Hot gravity filtration is commonly used to remove these impurities from a solution prior to recrystallization. Hot filtration is necessary for recrystallization when impurities exist in solution. Firstly, recrystallization requires a hot solution because the solution needs to be super saturation in order for crystals to form upon cooling. Secondly, the impurity may have different solubility in certain solvents than the compound to be crystallized. The idea is to choose a solvent that dissolves the compound to be crystallized upon heating, but the impurity doesn’t dissolve in the solvent at high temperatures. The impurity is then filtered out during the hot gravity filtration process.
The BCA assay is used to quantify protein concentration by using bicinchoninic acid to identify copper ions reduced by protein in a biuret reaction. The BCA protocol requires a working solution mixed with the sample; when protein is present, the reaction produces a purple color that absorbs light at 562 nm and is quantified with a spectrophotometer. The BCA assay is similar to other protein quantification assays such as Lowry or Bradford assays. However, the biuret reaction of the BCA assay occurs between the nitrogens on the peptide backbone and copper as well as nitrogens on the amino acid side chains. The fact that the peptide backbone participates in the reaction means that the BCA assay is more consistent between proteins and is less dependent upon amino acid composition. The BCA protocol is simple and quick. If the sample is heated to 37°C, then the incubation time is only 30 minutes, and the absorbance measurement takes only a few minutes. The BCA assay is an excellent method for quantifying total protein concentration after biomagnetic protein purification.