Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay

Several methods have been developed to quantify cytokines and chemokines in biological fluids and tissue culture samples, including bioassays, enzyme- linked immunosorbent assay (ELISA), intracellular staining, ribonuclease pr otection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we de scribe a new multiplexed assay, using the FlowMetrix(TM) system, that can q uantify multiple cytokines simultaneously in a small sample volume. This as say was found to be more accurate, sensitive and reproducible than the conv entional microtitre ELISA procedure. Furthermore, the time and cost involve d are comparable to, or less than, the ELISA. A key feature of the FlowMetr ix(TM) assay is its ability to multiplex: here, we show that this assay can accurately quantitate 15 cytokines in a 100 mu l sample volume while the s ame analysis by ELISA requires 1.5 ml (100 mu l for each cytokine assay). B y using this Flow Metrix(TM) assay, we could demonstrate that only T helper 1 (T(H)1)-deviated cells produce detectable levels of interleukin (IL)-2, while only T(H)2-deviated cells produce significant amounts of IL-4. Six ot her cytokines were produced by both T cell subsets, with the T(H)1 populati on producing more IL-3, granulocyte-monocyte colony stimulating factor (GM- CSF) and interferon (IFN)-gamma, and the T(H)2 population producing more IL -5, IL-10, and IL-13. Seven other cytokines were not produced in detectable amounts. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are av ailable. It will also provide a more complete picture of the plethora of cy tokines secreted during an immune response. (C) 1999 Elsevier Science B.V. All rights reserved.