Multiplexed bead assays have always been prone to aggregation, especially in the presence of complex biological fluids and matrices. The inherent properties of magnetic beads in regards to size, magnetism, and coatings (both polymer and conjugated protein) can lead the magnetic particles to clump or aggregate during the assay incubations, washings, and resuspensions.
Aggregation of beads causes free antigen to be trapped in the interstices and result in incomplete washing. The aggregated beads cannot be accurately quantified with the bead reader and will also cause clogging of the instrument. Thus, the presence of aggregates can cause variability in the assay itself leading to reproducibility issues, as well as low bead counts during the instrument reading.
DropArray is a miniaturized, wall-less 96-well plate that minimizes aggregation in bead-based assays and provides thorough washing and ∼100% recovery of beads, even in challenging viscous or lipid-laden samples.
In contrast, the use of standard 96-well microplates makes aggregation worse because of the large well size requiring more beads and the walls that provide an interface for trapping beads.
Data of a Milliplex assay performed at a medium-sized biotech company in the western U.S.
Data Produced with Milliplex® kit after Overnight sample incubation and image acquired on Incell 2000 at 10X.
A) A user observed fewer than 5 beads per analyte due to aggregation during incubation with sticky sample running over 2,000 beads per well on a standard microtiter plate.
B) In the same assay on a DropArray Plate, all the analytes showed more than 35 beads per analyte with minimal aggregation in spite of one-fifth the beads on a DropArray Plate, 400 beads per analyte vs. 2,000 beads per analyte on a standard microtiter plate.
The fact is, most of bead-assay suppliers that manufacture research immunoassays have information in their troubleshooting and support sections of manuals that describes how to deal with bead aggregation/clumping or low bead count. It is one of the major technical problems with bead-based assays. Bead aggregation is most common in biological fluids that have a complex matrix, such as plasma, synovial fluid, cerebrospinal fluid, etc.
The unique 2-dimensional incubation and laminar washing of beads on a wall-less plate over a magnet prevents clumping/aggregation that is common with biological fluids using the standard protocol of magnetic bead-based immunoassays (Figure 1). One of the most common mechanisms of bead aggregation is the matrix sample which functions as a glue to gather beads onto each other during an incubation step. On a DropArray plate, a plate is placed on top of a magnet during incubation as well as washing forcing beads to stay as a monolayer on a surface and therefore unable to aggregate. Thanks to a small volume of a drop on a DropArray plate, the binding equilibrium between beads and target analytes remains same as that of a conventional plate where beads float in a solution. The DropArray provides the ideal hybrid assay, combining both benefits of planar-array with solution bead arrays.
Figure 1. Comparison of bead-based assay on DropArray plate versus standard microtiter plate.