A magneto elisa is a combination of magnetic bead separation and Enzyme Linked ImmunoSorbent Assay (ELISA) for analyte detection. The magnetic bead separation helps to enrich the target population from complex media such as serum or whole blood prior to quantitative detection via ELISA. This works particularly well for cell separation and detection. One example where a magneto ELISA was used, was to detect CD4+ T-cells from whole blood of HIV patients. An accurate count of CD4+ T-cells is imperative in the treatment and management of HIV and detection of AIDS development.
Magnetic Bead Separation in magneto ELISA
Magnetic bead separation requires superparamagnetic nanoparticles. These particles are made of iron oxide, and are magnetized in the presence of a permanent magnet. Once magnetized, the particles move along a magnetic field gradient and collect near the permanent magnet source. Large magnetic beads are made of polymer embedded with many superparamagnetic nanoparticles. These beads are functionalized with surface molecules that can bind antibodies and other capture molecules. In the case of the CD4 magneto ELISA, the beads were functionalized with anti-CD3, which selectively binds T-cells. Once the T-cells were separated from whole blood, they were counted by ELISA.
ELISA principle and mechanism
The basic principle of an ELISA is the detection of an antibody-antigen binding event by measuring the light emitted by a chemical reaction enabled by enzymes conjugated to detection antibodies. The sandwich ELISA is commonly used to detect antigens. In continuation of the CD4 magneto ELISA example discussed above, the captured T-cells were also labeled with anti-CD4 antibodies conjugated with biotin. Streptavidin-HRP was added to the sample and it bound to the biotin anti-CD4 antibodies that were bound to the CD4+ T-cells. Horse Radish Peroxidase (HRP) is an enzyme commonly used in ELISA. In the presence of the substrate TMB (3,3’,5,5’-Tetramethylbenzidine) and hydrogen peroxide, HRP catalyzes the production of 450 nm light, which is quantified by a microplate reader and compared to a standard curve to provide a count of CD4+ T-cells.
Benefits of the magneto ELISA
An ELISA requires clean samples in order to achieve high assay sensitivity. Complex media such as whole blood or serum contains other proteins, lipids, carbohydrates, and other molecules that can interfere with antibody-antigen binding or can non-selectively bind to the plate or other surfaces; this causes high background interference and can decrease the sensitivity of the ELISA.
Immunomagnetic cell separation is a rapid and efficient method of enriching target cells and washing out unwanted debris before further analysis. It is important to choose a well-designed magnetic separation rack when performing magnetic cell separation because poorly designed racks can cause unwanted cell lysis or clumping or magnetic beads. A homogeneous magnetic force over the entire working volume is the key to a rapid, efficient, and gentle magnetic cell separation process. The cleaner the magnetic separation, the easier it is to perform further analysis on your target.
Carinelli, S.; Xufré, C.; Alegret, S.; Martí, M.; Pividori, M.I. Talanta 2016. 160, 36-45.