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.
Magnetic bead antibody binding and attachment chemistry
There are two main methods of binding antibodies to magnetic beads: covalent bonds and noncovalent bonds. Examples of non-covalent attachment methods are streptavidin-biotin affinity and protein A/G. Magnetic beads of both types are available. The downfall of streptavidin beads is that the antibody must be pre-conjugated with biotin before incubating with beads. Protein A/G is an attractive option because these proteins bind most IgG antibodies.
Covalent attachment chemistry is another nice method for creating magnetic bead antibody conjugates. The benefit of these methods is that the attachment is irreversible. When working with antibodies or any protein, the most common attachment point is amine groups. Carboxyl and sulfhydryl groups are also used, but less often. Here are four of the most common magnetic bead coatings:
- tosyl groups-hydrophobic bead that binds amine and sulfhydryl groups at neutral pH and 37ºC
- carboxylic acid-hydrophilic bead that binds amine groups at pH 5-6 at room temperature
- amine-hydrophilic bead that binds aldehydes at neutral to high pH at room temperature
- epoxy-hydrophilic bead that binds amine and sulfhydryl groups at neutral pH at a range of temperatures
Uses for magnetic beads antibody conjugation
The most common uses for antibody-conjugated magnetic beads include magnetic activated cell sorting and immunoprecipitation. Magnetic cell sorting requires magnetic beads conjugated with antibodies that recognize specific cell surface receptors. These receptors are chosen because they are unique to the target cell type. As an example, if the target cell is a T-cell, then the magnetic beads will have antibodies toward CD4 or CD8. This would be a fairly general enrichment of T-cells. From here, additional magnetic sorting rounds can be completed with more specific antibodies to enrich a particular subpopulation of T-cells to match the goals of the study. A similar method is used in immunoprecipitation, but in this case the goal is to enrich proteins, not whole cells. The size of the magnetic beads will likely be different between magnetic activated cell sorting and immunoprecipitation protocols, but the chemistry used to attach antibodies will be similar. The commercial availability of magnetic beads in a range of sizes and surface coatings makes these types of experiments easy to design and perform.
