July 1, 2009 (Vol. 29, No. 13)

Availability of Suitable Washing Equipment Aids in Obtaining Rapid and Accurate Results

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.

Comparison of Washing Methods

Multichannel pipettes, versatile and ubiquitous in almost every laboratory, may be used for any aspirate and dispense step in the magnetic bead-based assay process when used along with a permanent magnetic support, although by design their use is ultimately inappropriate for these tasks.

Consistency is highly dependent on the operator and difficult to reproduce from plate-to-plate or even from one procedural step to another. Tip depth and force used are also operator-dependent. Incorrect tip depth can lead to residual liquid, unwanted aspiration of beads and damage to the microwell surface, while incorrect aspirate/dispense force can contribute to aspiration of beads or splashing and aerosol creation.

A vacuum manifold, considered the traditional standard for many bead-based assays, may be used if the assay is performed in a filter-bottom microplate. Per each wash step, the filter-bottom microplate is sealed in an airtight chamber, and a vacuum is applied, drawing out the liquid. Additional reagents are dispensed via an additional device or instrument.

Vacuum manifolds have been used for decades, and their simplicity and low relative cost are attractive. It is difficult, however, to regulate vacuum pressure from well-to-well and plate-to-plate—too low, and residual liquid remains in the well; too high, and beads may become trapped within the filter membrane. Once removed from the chamber, residual liquid on the underside of the microplates must be blotted—a messy step that is difficult to build into automated systems. Finally, vacuum manifolds are not adaptable to various fluid viscosities, and their inherent vibrations may also contribute to blockages in the filter membrane.

Microplate washers are readily integrated into automated systems, and those equipped with magnets such as the ELx405™ Magnetic Bead Washer from BioTek Instruments can easily be integrated into magnetic bead assay procedures (Table). These washers significantly reduce human error and produce consistent results through every well and microplate. Gentle yet thorough aspiration and dispensing are highly repeatable and minimize bead loss through numerous wash cycles and assay parameters.

Comparison of magnetic bead assay wash methods

The ELx405 Magnetic Bead Washer (Figure 1) offers high bead recovery, allowing for better sensitivity when the beads are read on a flow cytometer. Other wash methods are significantly less efficient, averaging 60–80% or less bead recovery (data not shown), which decreases sensitivity and may be problematic when working with rare or expensive compounds. Integrated software, controlled from the instrument keypad or remote PC interface, allows for adjustment of all parameters including independent control of microplate position during aspiration/dispense cycles, fully programmable fluid volumes and flow rates, shaking, and soak time. A cavity within the microplate carrier holds a removable magnet so that the user can perform magnetic bead and traditional assays on the same instrument. This specialized magnet generates a high magnetic field and is optimized for magnetic bead assays, for rapid and uniform separation in each microplate well.

By reducing human sources of error, and eliminating variability and membrane clogging experienced with vacuum manifolds, the automated washer provides improved consistency. The automated microplate washer delivers consistently lower CV levels than other wash methods and demonstrates no high CV data points consistent with filter clogging or human error (Figure 2).

Bead-based assays continue to increase in popularity, with magnetic-bead assays among the most precise and easy to use and automate. However, use of these assays can still be impaired by lack of suitable washing equipment. An automated microplate washer provides high bead recovery, sample-to-sample consistency, and a reduction in direct operator involvement, for rapid and accurate results.

Figure 1. Percent of magnetic beads recovered after numerous wash cycles and variable assay parameters using an automated system including the ELx405 Magnetic Bead Washer.

Figure 2. Variability in replicate data comparing three wash techniques

Jason Greene ([email protected]) is product manager at BioTek Instruments. Web: www.biotek.com.

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