Enzyme-linked immunosorbent assays (ELISAs) continue to evolve to meet increased user demands. One such advancement stemming from traditional ELISA technology is the use of microspheres (beads) as a solid support matrix in conjunction with microplates. Bead-based microplate assays provide greater surface area for binding with lower background signal than traditional ELISAs.
Additionally, beads can be coded and used in multiplex assays, which measure multiple analytes in a single microplate well to reduce overall cost and time per assay. Automating the assay serves to boost sample throughput further while reducing potential sources of error.
Most bead-based multiplexed assays are provided as panels. Cytokine panels, for example, are offered by many companies. Cytokine panels contain up to approximately 30 different cytokines that can be measured from a sample in a single well. Samples can be body fluids, cell supernatants, or cell lysates. Typically, there are sample matrix interferents and excess reagents that must be removed to allow for accurate, precise measurements of the plexed analyte.
With most bead types (e.g., polystyrene, latex), this step incorporates washing with a filter-bottom microplate under vacuum. Unfortunately, vacuum filter-based washing is prone to individual wells clogging, which tends to reduce precision and the method is not automation friendly. Newer magnetic beads are of particular interest as washing is simply done with a microplate under a strong magnetic field, which alleviates problems with precision and automation.
Magnetic beads used in almost all multiplexed assays are made in a similar fashion to standard beads such that they can be coded, but a partial coating of iron oxide is added. During wash steps, an external magnet is applied to the microplate, thus immobilizing the beads. The partial coating of iron oxide requires a strong magnetic field to ensure that beads are not lost during wash steps.
Iron oxide within the bead is often superparamagnetic, which means that once removed from an external magnetic field the beads will not retain residual magnetism that may lead to clumping and a subsequent decrease in assay efficiency. A small residual volume of wash buffer is left behind after each wash cycle to ensure good bead recovery. The process effectively emulates a standard ELISA protocol where analyte is captured at the bottom of a microplate well and successive wash cycles remove unwanted interferents. With the exception of the magnet, all automation and liquid-handing instrumentation common to ELISAs may be used.