Isolation and detection of a target molecule in cell therapeutics from a sample with high background debris or unwanted molecules is a challenging task. Immunomagnetic-based separation is the most feasible technique to overcome the problems that come with the separation of cells and biomolecules from a complex matrix.
The main advantage of immune-based separation is the high specificity of the technique. Antibodies are generated by the adaptive immune system to perfectly identify the epitope of the antigen that stimulated the immune response. While off-target effects do exist they are fairly rare, and most antibodies demonstrate highly specific affinity for their target antigen. This means that the background and false positives of immune-based separation is quite low. When paired with biomagnetic separation ( magnetic activated cell sorting) the result is a highly efficient and rapid method for concentration and enrichment of target cells from a complex matrix. This technique is highly adaptable, and is most commonly used with body fluid or cultured cell samples that include a large diversity of material.
The basic principle of immunomagnetic separation involves the attachment of small magnetic beads/particles to target cells via specific antigen-antibody attraction. The beads, pre-coated with a specific ligand, are added to a mixed suspension containing the desired cells. These magnetic beads bind to their target via specific antibody/antigen recognition to form a complex. When a magnetic field is used, those cells that have beads attached will be attracted to the magnet and are removed from the suspension. In this way, it allows labeled cells to be separated from unwanted unlabeled cells and molecules. The cell separation can be achieved using flow cytometry or magnetic-activated cell sorting.
These beads are super-paramagnetic, mono-dispersed, inert, polystyrene shell/nanobeads-coated with a specific ligand compatible with the target. The beads used for immunomagnetic separation can be coated with a variety of capture molecules:
- primary antibodies; specific for the cell type,
- species-specific secondary antibodies,
- lectins or enzymes.
- streptavidin (used in conjunction with biotinylated primary or secondary antibodies).
Another option can be that the cells are labelled with primary antibody, and then species-specific secondary antibody-coated beads are added to the suspension. However, the required strength of the magnetic field always depends upon the size of the beads.
Immuno magnetic technique is especially useful in diagnostic microbiology and immunology for the concentration, isolation and/or purification of various microorganisms including fungal/bacterial cells or spores, parasites, cellular and sub-cellular material like viruses, biomolecules like proteins, and nucleic acid and cancer cell products. By this technique, a considerable amount of time can be saved, because it excludes the standard enrichment that is required using conventional microbiological methods.
The enriched and concentrated samples are then suitable for further analysis via polymerase chain reaction or other methods where a clean sample is required.
Applications of Immunomagnetic cell Separation
Immunomagnetic cell separation is often used to determine pathogenic contamination of food. Small concentrations of pathogens in an otherwise normal food supply can cause devastating effects, and immunomagnetic cell separation can be used to test for pathogen contamination.
Determining antibodies in human blood is also a feasible application. Antibodies tend to be in low concentrations compared to the large amount of hemoglobin in blood, and the specificity of antigen-antibody interactions allows for low concentrations to be identified. This was one of the first methods used to confirm antibodies developed against the COVID-19 virus.
A critical aspect, often overlooked, on immunomagnetic cell separation are the devices used during the process. For very small volumes, first tries are usually done with simple magnets. However, using this technique, the magnetic force changes very quickly with the distance, then small variations in the position can lead to great variability in the efficiency. When the problems become apparent, the tendency is to point to the magnetic beads, ligands or the buffer composition, but not to try to properly specify the magnetic separation protocol. To do so, the right approach is to use a constant magnetic force device, like modern biomagnetic separation systems (link to Sepmag LAB webpage https://www.sepmag.eu/separators-for-rd/sepmag-lab).
That allows testing the right magnetic force for the application -different devices with different forces can be tested- and specify the right magnetic force for the application. As the separation speed is directly proportional to the magnetic force, scaling up the process would just need to use the same value of the force and increase the separation time proportionally to the tube diameter.
- Magnetic Beads and Biomagnetic separation
- Applications of Biomagnetic Separation
- Magneticbeads for sensitive detecting plant vacuolarprocessing enzyme activity