Multiplexing technologies have emerged as a powerful strategy to acquire more data from a given sample. Essentially these technologies allow you to detect more than one factor simultaneously, thus saving you time and money in both reagent use and sample preparation.
BD Biosciences Pharmingen BD Cytometric Bead Array is a multiplexed flow cytometric bead assay that is used to quanitate soluble analytes. The system is comprised of 6 spectrally unique beads that are coated with capture antibodies to detect cytokines from cell supernatants or serum samples. Each bead has a unique spectral emission that is detected in the FL3 channel of a standard flow cytometer. The beads are incubated with the sample containing the analytes of interest. The soluble analytes are captured by the bound antibody. A second antibody that is conjugated to phycoerythrin (PE) detects bound analyte. The assay system is a basic sandwich ELISA on a bead format using optimally paired antibodies. Two-color detection is made possible on any flow cytometer with a 488 nM laser, including the FacsCalibur and the FacScan.
The advantage of the BD CBA system is that it allows you to detect and quantify multiple analytes from small sample volumes. Unlike other multiplex bead formats, it allows for detection on any given flow cytometer, so for most immunology laboratories, there is no additional equipment necessary. I have primarily used both the human and mouse cytokine detection kits for detection of IL-2, IL-4, IL-6 (or IL-5), IL-10, TNFa, IFNg or the inflammation kits that detect IL-8, IL-1b, IL-6, IL-10, TNF, IL-12. The detection range is very good, with high sensitivity and low cross reactivity. Standard curves are generated using recombinant cytokines. I have used anywhere from 15-50 uL of supernatants for T cell functional assays and observed good cytokine detection.
Pre-configured kits include multi-cytokine detection for both mouse and human, a human anaphylactic kit, human chemokine detection kits, mouse isotyping kits, and kits for detection of signaling molecules that include phosphorylated stat1 and apoptosis signaling proteins. Each kit us useful for 50 assays.
The BD CBA system also provides easy to use software analysis package that simplifies the data processing, although the analyis can be performed without the software. This can be extremely helpful for individuals not accustomed to this type of data collection. In addition, the product manual is extensive and gives details on to how to set up the bead collection. Although the cost of the kits might be discouraging, when comparing the cost of the CBA to that of 6 ELISAs, the savings are readily apparent, not to mention the time saved in obtaining multiple data points from the same sample.
Overall, the CBA kit is convenient, easy to use, and extremely suited for detecting multiple analytes from small volumes. It’s also a flow cytometric based kit, so it doesn’t require the purchase of extra equipment. The kit has great potential for the BD Facs Bioanalyzer or any multi-well autosampler. In addition, BD representatives are very knowledgeable and can assist in the setup of the assay. Once you start multiplexing, you’ll wonder why you even bothered with single point ELISA systems.
Omar Perez, Ph.D.
Post-Doctoral Fellow
Nolan Lab
Stanford University