The principle behind multiplex assays depends on the use of uniquely colour-coded polystyrene beads (microspheres), that allows the identification of multiple targets in a single sample. In Bender MedSystems’ FlowCytomix kits, there are two sets of microspheres – each is a different size and made up of five bead populations that internally contain different fluorescent dyes. This allows the simultaneous quantification of ten cytokines in a small sample volume. A monoclonal antibody directed against the required cytokine is coated to microspheres. The conjugated beads are allowed to react with the sample or with a standard solution containing a known amount of cytokine. Then a biotinylated detection antibody specific for a different epitope on the cytokine is added to form a sandwich of antibodies around the cytokine. The biotinylated detection antibody binds to streptavidin and, after the addition of a streptavidin-phycoerythrin complex, the emitted fluorescent signals are detected using a flow cytometer.
The Th1/Th2 10plex Kit II allows a quantitative detection of 10 human cytokines including IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IFN-g and TNF-a in as small as 25ul of cell culture supernatants, plasma, serum, whole blood or any other biological sample. I have used this kit to measure these cytokines in cell culture supernatants. The protocol is easy to follow and can be adjusted in cases where less than ten cytokines are being measured. Two sets of standards are supplied with the kit, and from my experience each set can be frozen at -20oC and used once more, therefore, each kit can be run four different times if someone doesn’t have many samples to run at once. Standards, beads, biotin-conjugate and streptavidin-PE should be prepared prior to performing the assay. Test procedures can be performed in flow cytometer tubes directly or in a special filter plate, which requires a vacuum filtration manifold. The company recommends storing samples for a maximum of 24 hours but from my experience they can be stored for up to one week at 2 – 8oC protected from light without significant lose of their fluorescence intensities. Samples are analyzed on a flow cytometer after adjusting voltage of FL-2/FL-3(-4) and compensation using setup beads provided with the kit and the highest standard. I must stress here on the importance of an accurate compensation between FL-2 and FL-3, which is a critical issue and should be adjusted carefully. Data analysis software, called “FlowCytomixPro 1.0”, is provided with the kit or can be downloaded free from the company’s website.
In summary, detection of several cytokines in one sample can save a lot of effort and time. This type of analysis is particularly useful when the volume of test samples is very limited. One of the most important advantages of using this system is that no special instrument required to run these beads and the most commonly used flow cytometers (e.g. FACScan or FACSCalibur) are required for running samples.
Eyad Elkord, Ph.D.
Post-Doctoral Fellow
Paterson Institute for Cancer Research
The University of Manchester, UK