The Cytometric Bead Array (CBA) from BD Pharmingen is a flow cytometric based assay for the detection of human cytokines from cell supernatants or serum samples. The kit uses a set of beads that have been coated with cytokine specific antibodies which serves as a capture surface for cytokines. Each cytokine specific set of beads is assigned a discrete fluorescence intensity that can be resolved on the FL-3 channel of a flow cytometer. The beads are incubated with the sample of interest, washed, and then incubated with a second cytokine antibody that is PE labeled. In this manner, a bead that is positive for a cytokine will stain both PE and FL-3 positive, while beads that are specific for cytokines that are not present will stain only FL-3 positive. A mixture of the capture beads and PE labeled secondary antibodies allow for the detection of multiple cytokines/proteins from a single, small sample volume. The kit comes with an extensive manual that describes the exact set up for the acquisition of the data and how to run the standards supplied by BD Pharmingen in order to calibrate the system. Both 6 bead and 8 bead kits are available (50 uses per kit) and new cytokines may be available in the future (based on both bead fluorescence intensity and bead size). BD Pharmingen also suggests purchasing the analysis software specifically designed to analyze data acquired by CBA assays.
I was using this kit to analyze T-cell supernatants in order to distinguish between TH1 and TH2 reactions. To do this, I used the 6 bead kit, which includes beads specific for IFNg, TNFa, IL-2, IL-10, IL-4, IL-5. I was please to find that it worked well from the first time I tried it. Initially I ran several dilutions of an IFNg standard as my own control and different dilutions of a T-cell mitogen. Analysis showed a perfect dose response to each and the results were clear - even slight differences in concentration were easy to compare. If the CellQuest template set up suggested by the manual is used, positive and negative results are obvious during flow cytometric acquisition. The kit claims it has high sensitivity and I have not seen anything to dispute that. I typically run supernatants from 24 hour stimulated T-cells and use approximately 50 ul with the CBA beads and can see good cytokine production. The kit is fast and easy to use and by following the booklet the acquisition set up is relatively easy as well.
The advantage of using the CBA assay is that it is an accurate and sensitive method for measuring cytokine levels. In addition, it requires small volumes for analysis. The 6 and 8 bead kits are very convenient in that they allow for simultaneous detection of multiple cytokines, further reducing the volume needed per assay and number of samples for analysis. This method is also quantitative for each cytokine. A big convenience is that you can freeze samples and run the CBA when ready. The kit itself requires only a few hours to run.
One of the down-sides of the kit is its price. It is expensive and they also suggest purchasing the software. The software may not be necessary since it is an add on to Microsoft’s Excel program. If the kit were only going to be used a limited number of times, it is not worth purchasing the software. Also, it is important to be aware that the kit does not have the same sensitivity levels for all cytokines, though the manual notes the sensitivity for each cytokine included. One annoying problem is that the standards must be run each time the kit is used in order to quantitate the results. This unfortunately means the waste of a significant part of the kit to running standards and therefore a potential waste of money. One of the ways to avoid this problem is to freeze multiple samples and run as many together as possible. A BD representative also suggested testing an experimental positive control and see if it worked before running the entire set of samples and standards, making sure the experiment worked before wasting the entire kit.
Overall, the CBA kit is a very convenient way for quantitating multiple cytokines from a small volume. It has several advantages over using cytokine ELISA kits- less volume and sample required, because it simultaneously tests for multiple cytokines, and less time to run the assay. And though the CBA kit is expensive, it is probably cheaper than buying 6 to 8 separate ELISA kits. The analysis software is worthwhile if the kit is used routinely, but a BD representative may needed to set it up. I would recommend this kit for anyone needing to test for multiple cytokines and have limited sample volumes. I feel it is accurate as well as easy to use.
Amy Hobeika, Ph.D.
Post Doctoral Associate
Duke University Medical Center