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.
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.