The Bio Rad Bio-Plex System is a compact, simple, highly flexible platform for a wide range of bead-based assays. It combines the concept of flow cytometry with dual red and green lasers for simultaneous bead and reporter detection with up to 100 uniquely identifiable fluorescently labeled microspheres. This combination enables bio-molecule analysis in a highly multiplexed format that can save time and money while conserving limited or precious samples. Individual microspheres (or beads) can be complexed with any protein, peptide or nucleic acid. The software is easy to use and directs the user at every step with pop-up windows that give clear, concise directions.
Assay kits are currently available from Bio Rad for cytokine and phosphoprotein detection and analysis, as well as, coupling kits for designing your own assay. Analysis is performed in liquid phase with minimal wash steps utilizing analytes labeled fluorescently with phycoerythrin (R-PE), AlexaFluor dyes or Cy3. Beads complexed with biomolecules and reporter analytes are shuttled through the detector individually, while the red laser identifies the bead and the green laser reads the fluorescence intensity of the reporter molecule associated with that bead. Since fluorescence signal is only read in tandem with bead signal, background is greatly minimized. The reader takes fluorescence measurements for a minimum of 100 beads of each type in any given well and reports the average signal intensity for that bead.
Our lab uses the Bio-Plex system for determining serum antibody reactivity, antibody specificity and nucleic acid-binding assays. We combine the Bio-Plex reader with LiquiChip beads available from Qiagen. LiquiChip Ni-NTA beads allow us to complex beads with any 6xHis-tagged protein and the LiquiChip Carboxy beads allow us to complex proteins without His-tags, as well as, crude cell lysates. Even without washing the beads after protein binding, we do not see any “crosstalk” or contamination when different bead/protein complexes are mixed together for multiplex assays. An entire 96-well plate of multiplexed assays can be read within 1-2 hours, including reader calibration and post-analysis cleansing. We calibrate before every set of assays and see no more than 10-15% variation between duplicate analyses, but since signal intensities are high and background so low this variability has been quite acceptable. Our main frustration has been the inability to access the signal intensities for the individual bead readings within a single multiplexed assay well. Since at least 100 readings are taken for each bead-protein complex in an individual reaction well, it would be nice to do statistics on the readings within that well. As it is, the only statistics we can perform are between duplicate assay wells. Though the average of 100 or more data points is significant, it would be comforting to know internal variability.
The Bio Rad Bio-Plex system has been a reliable and user-friendly addition to our lab repertoire. We highly recommend it based on the flexible build-your-own-assay format. Results are obtained with minimal hands-on time and minimal sample manipulation in highly multiplexed format. The ability to perform statistical analysis on readings in a single assay well would be a great improvement for use with precious samples, but isn’t too much of a problem if you can run multiple, identical multiplexed assays in the same plate.
Becky Bundy, Ph.D.
Post Doctoral Associate
Center for Tropical and Emerging Global Disease
University of Georgia