The enzyme linked immunosorbent assay (ELISA) is a gold standard method for protein and antibody detection. It is routinely used for clinical lab work and is often used in research and development. The assay is based on the lock-and-key specificity between an antibody-antigen pair. Antibodies are naturally made by the adaptive immune system to recognize a specific piece of an antigenic protein. This is what allows the body to identify and fight invading pathogens so effectively. Since antibodies have such a fundamental role in most biosensing and diagnostic systems it is very easy to purchase them or have them custom-made to recognize a protein of interest. The ELISA assay is performed in 96- or 384-well plates that are treated to favorably adsorb antigen or antibody depending on the type of ELISA being performed. The ELISA assay is built up from the bottom of each well; it doesn’t float freely in the well. As a result, the unbound proteins are easily washed away and only the target remains to be quantified.
The concept of an antigen and antibody pair is central to modern medicine and biotechnology. These proteins match like a lock and key, with equisite specificity. The interactions are non-covalent, but have equilibrium constants ranging from 105 to 1012 M-1 Antibodies and antigens are proteins: polypeptide chains of amino acids. The IgG antibody is composed of four polypeptide chains, two heavy and two light, organized into a ‘Y’ shape. The base of the Y is called the Fc region, while the two tips are known as the Fab region.
Chemiluminescent immunoassays (CLIAs) are excellent assays for high-throughput, low analyte concentration and time sensitive testing and isolation. Using coated magnetic beads, such as streptavidin beads, as the reagent in a CLIA is an easy and established technique favored among many clinical scientists.
When scaling up a process using a traditional magnetic separation rack, the percentage of bead and biomolecule losses significantly increases with an increase in volume. One way of dealing with this problem is by applying a higher force at longer distances. But for this to work, you must apply this greater force without increasing the forces in the retention area during the magnetic separation process, in order to avoid irreversible aggregation.
If one wants to scale up production from small lab lots to full-scale large lots, a non-homogenous magnetic separation process will result in lot-to-lot inconsistencies. Homogenous biomagnetic separation conditions, however, guarantee consistent results regardless of production scale.
Due to the inherent properties of classic non-homogenous biomagnetic separators, beads can aggregate during the magnetic separation process. When this happens, technicians try to resolve the magnetic beads separation problem by using special resuspension techniques like the sonication method. But problems with resuspension can ultimately lead to end-product variability, especially if aggregation is not detected early.
In-lot consistency is the key to reproducibility at the level of a kit. Unfortunately, in non-homogenous systems irreversible aggregation is one of the main sources of in-lot variability. If all of the beads are exposed to the same force as they are in homogenous magnetic systems, the risk of aggregation is greatly reduced. Because of this, it is important to know how to avoid irreversible aggregation problems during a magnetic separation process.
A recognized problem in the biomagnetic separation industry is that when one increases the batch size to scale up production of magnetic beads, the magnetic separation process time increases unproportionally to the increase in volume if one is working with standard magnetic separation devices.
When using biomagnetic separation, in order to ensure the consistency of the resulting product and the process itself, there must be some sort of validation procedure. Validation should be consistent within a given lot, from lot to lot and also when the process is scaled up. The validation procedure should optimally be related to the conditions of magnetic bead separation and not be dependent on any specific device that generates the magnetic field.