Protein A beads like ELISA, Immunoprecipitation, antibody purification, and multiplex assays require the attachment of specific antibodies to a solid support such as a column, polystyrene plate, agarose bead, or superparamagnetic nanoparticle. There are a number of ways that antibodies are attached to solid supports. Some of these include:
Fluorescent microspheres are small polymers embedded with fluorescent dye. They are a useful tool for medical imaging because they are non-toxic and non-biologically reactive when used as directed. Fluorescent microspheres are also useful in research laboratories as markers for fluorescent microscopy and as standards for flow cytometry fluorescent cell sorting. The main benefit of using a polymer microsphere embedded with fluorescent dye rather than using the dye alone, is two-fold: the matrix protects the dye from photobleaching, and the microsphere concentrates the dye leading to a more robust fluorescent signal.
The structure of the most commonly known antibody class (IgG) was a mystery until 1959, when it was elucidated by Edelman and Porter . The duo approached the question from two very different directions, but they were both awarded the 1972 Nobel Prize in Physiology or Medicine for their groundbreaking work. Gerald Edelman said that he was, “fascinated by the specificity of antigen recognition by antibodies,” and hoped that, “by doing the primary structure of antibody molecules, the basis of their specificity would be revealed.” Indeed, it was.
The goal of the series of posts from the last weeks was to review the state-of-the-art of magnetic beads coatings. The contributors have reviewed the classical surfaces, but also the new approaches to improve and simplify the process. Last but not least, the physical aspects of the magnetic beads and the separation process were discussed, as they have a critical impact on the success of the coating process.
Coating your magnetic beads with biomarkers is the most critical step during the development and production of Chemiluminescence Immunoassays (CLIA). Attaching the antibody (or any other protein) to the bead’s surface requires incubating both materials together, using the right buffer and temperature, gently mix and homogenize the suspension. Once the process is completed, it is necessary to separate the solid phase (the magnetic beads with the attached biomolecule) from the rest of the suspension and, once washed, re-suspend the reagent in a new buffer for avoid biomarker reaction and beads aggregation.
During protein (or other kind of molecules) coating onto magnetic particles, there are two main parameters that govern the success of the process: the physical and chemical properties of the protein itself and the magnetic particle dispersions. For this reason, the correct selection of these components is the key for an excellent coating. In this article the importance of physical properties of magnetic dispersions is discussed.
Magnetic beads are available with a large variety of surface coatings. One of the coatings are the Tosyl activated beads. This post is describing the handling and advantages of the use of Tosyl activated magnetic beads in chemiluminescent immunoassays.
The use of magnetic beads in IVD is not new. Recent developments –as the described in the next chapters- promise easier and better coating procedures where the orientation and the availability of the captured molecule can be controlled. However, most of the current applications are still using the classical surfaces.
Designing binding surfaces with optimal ligand (e.g. antibody, antigen or protein) functionality is required for ultra sensitive assays. However, classical solid phase chemistry approaches for conjugating or binding ligands to surfaces do not control the density or parking area of the ligand, nor do they provide control over ligand conformation and orientation.
KODE™ Technology is based on novel water-dispersible self-assembling molecules, called a function-spacer-lipids or KODE™ constructs (Figure 1) that are able to coat virtually any biological or non-biological surface with almost any biological or non-biological material [1-10]. The primary coating method of live cells, organisms, bacteria and viruses or solid surfaces (glass, metals, plastics, etc.) is achieved by simple contact with a solution containing one or more FSL KODE™ constructs. Upon contact the FSLs spontaneously and harmlessly create a stable and novel surface coating. Essentially the spontaneous self-assembling process is driven by the need of the constructs to “exclude water”. Because the constructs are able to bind to virtually any surface, be it hydrophobic or hydrophilic the mechanisms of action are multiple and complex and include hydrophobic interactions (via lipid tail), hydrophilic interactions (via the head group and spacer), micelle entrapment, encapsulation, bi/multi layer assembly, and other factors such as hydrogen bonding, van der Waals forces, electrostatic and ionic interactions and combinations of all the above on complex surfaces.
