Cell Immunomagnetic separation protocol

Cell Immunomagnetic separation is a technique for isolating specific cell populations or diagnosing pathogens in clinical samples. The technique is based on two main components: antibody-antigen specificity and superparamagnetic beads. These components can be tuned to the experimental goal, and can produce high yields and highly enriched targets when used properly. The most common targets are cells that display unique identifying surface antigens. The immunomagnetic separation protocol follows these general steps

1. Identify the target and characterize the unique surface antigens.
2. Choose a superparamagnetic bead that can be modified and functionalized with an antibody specific to the antigen on the target.
3. Incubate the functionalized superparamagnetic beads with the sample.
4. Isolate the target through magnetic separation.

Superparamagnetic beads

Superparamagnetic beads are nanoparticles of iron oxide. The nanometer size imparts these particles with superparamagnetic behavior, meaning that they are not innately magnetic, but become magnetized when placed into a magnetic field.  This property is useful for magnetic separation because the movement of the particles can be manipulated for controlled collection and isolation.

These beads can be functionalized with surface proteins such as proteins A or G that bind antibodies. They can also be biotinylated for the attachment of antibodies through biotin-streptavidin affinity. Many of these beads are commercially available with common antibodies pre-attached. Additionally, there are full kits available for the immunomagnetic separation of common cell types. 

Positive vs. negative immunomagnetic separation

There are two general strategies for immunomagnetic separation: positive separation, and negative separation. In positive separation, the superparamagnetic beads are functionalized with an antibody that specifically binds to the target cell to form a bead-target conjugate. That conjugate is then isolated by magnetic separation and the remaining contaminating solution is washed away. Positive separation is useful when the target has a unique surface antigen and the chance of non-specific binding is low.

In negative separation, the superparamagnetic beads are functionalized with a cocktail of antibodies that specifically bind to all other cell types in the solution except for the target cell. These other cell types are removed by magnetic separation, and the remaining solution is enriched for the target cell. Negative separation is beneficial when a specific surface antigen for the target cell has not been identified or when the presence of magnetic particles on the target cell could hinder downstream procedures.

Magnetic separation rack

Traditional block magnetic separation consists of a permanent magnetic block placed next to the container holding the sample. The magnetized superparamagnetic particles move toward the magnet and collect at the side of the container. The downfall of this setup is that the cells farthest from the magnet may not feel a sufficient magnetic force, if any, and will not be collected, while the cells closest to the magnet may feel a great force that leads to cell membrane damage.  Advanced biomagnetic separation racks avoid this problem by ensuring that all cells experience equivalent force throughout the working volume. The magnetic force does not vary with distance in advanced separation racks, which leads to higher yield and greater purity for immunomagnetic separation protocols.

Designing an immunomagnetic separation protocol

When designing an immunomagnetic separation protocol it is important to address the type of bead used, the bead surface functionalization strategy, and the type of separation rack employed. When all of these components are properly tuned to the experimental goal it is possible to achieve accurate enrichment of the target in a short separation time.