Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay

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Abstract

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 protection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we describe a new multiplexed assay, using the FlowMetrix™ system, that can quantify multiple cytokines simultaneously in a small sample volume. This assay was found to be more accurate, sensitive and reproducible than the conventional microtitre ELISA procedure. Furthermore, the time and cost involved are comparable to, or less than, the ELISA. A key feature of the FlowMetrix™ assay is its ability to multiplex: here, we show that this assay can accurately quantitate 15 cytokines in a 100 μl sample volume while the same analysis by ELISA requires 1.5 ml (100 μl for each cytokine assay). By using this Flow Metrix™ assay, we could demonstrate that only T helper 1 (TH1)-deviated cells produce detectable levels of interleukin (IL)-2, while only TH2-deviated cells produce significant amounts of IL-4. Six other cytokines were produced by both T cell subsets, with the TH1 population producing more IL-3, granulocyte–monocyte colony stimulating factor (GM-CSF) and interferon (IFN)-γ, and the TH2 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 available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response.

Introduction

Cytokines play a central role in the regulation of hematopoiesis; mediating the differentiation, migration, activation and proliferation of phenotypically diverse cells (Mosmann and Coffman, 1989; O'Garra and Murphy, 1994; Constant and Bottomly, 1997). Many cytokines are pleiotropic, possess overlapping functions and regulate the production of other cytokines. Consequently, the make-up of the cytokine milieu is often of greater importance than the actions of a single cytokine. The analysis and quantitation of cytokines in biological fluids and tissue culture supernatant has become a widely-used procedure in research and clinical laboratories, and is clearly important in furthering our understanding of many immunological functions.

Enzyme-linked immunosorbent assays (ELISA) are the most popular methods for quantitating secreted cytokines due to their high specificity and sensitivity (Mosmann and Fong, 1989; Hsieh et al., 1992). These assays involve sandwiching a cytokine between two cytokine-specific Abs that bind to mutually exclusive sites on the cytokine. The first `capture' Ab tethers the cytokine to a solid support. The second `detection' Ab is biotinylated in order to link the captured cytokine to a common detection system. Many cytokine-specific Ab pairs are now commercially available. The sensitivity of this approach has been significantly enhanced with the development of the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) using europium-labeled antibody (Ogata et al., 1992). However, ELISA, DELFIA and bioassays are limited to measuring a single cytokine per assay. Simultaneous detection of a limited number of cytokines can be performed by detecting cytokine mRNA using RT-polymerase chain reaction (PCR), northern analysis, ribonuclease protection assay (RPA) or in situ hybridization, or by detecting intracellular cytokine protein using flow cytometry (Sander et al., 1993; Bucy et al., 1995; Naylor et al., 1995; Openshaw et al., 1995). However, none of these latter techniques quantifies the amount of secreted protein.

The FlowMetrix™ System (Luminex, Austin, TX) uses microspheres as the solid support for conventional immunosorbent assays with a green-fluorescent reporter dye (Fulton et al., 1997; Keij and Steinkamp, 1998; Kettman et al., 1998). The capture antibody of each immunosorbent assay is coupled to one of 64 different microsphere bead sets that are currently available. Each bead set is comprised of microspheres manufactured with a uniform, distinctive proportion of red and orange fluorescent dyes. Once coupled, microspheres from different bead sets can be pooled together for the assay and separated later during data acquisition. The immunosorbent assay results in a variable amount of the green reporter dye, proportional to the amount of analyte, bound to the surface of each microsphere. Data are acquired on a conventional flow cytometer (FACScan or FACSCalibur) connected to a PC equipped with a special digital signal processing board and control software that runs within Windows 95. The flow cytometer quantitates the green, orange and red fluorescence of each microsphere using the FL1, FL2 and FL3 detectors, respectively. The software uses the orange and red fluorescence data to separate the pool of microspheres into individual bead sets and presents the average amount of green fluorescence for each bead set. As the individual bead sets can be separated by a flow cytometer, many assays can be performed simultaneously, thus achieving multiplexed quantitation of multiple analytes in a single sample. Here, we report the use of this system for the simultaneous quantitation of 15 secreted cytokines in a small sample volume (100 μl). Its sensitivity, reliability and accuracy are compared to a standard microtitre ELISA procedure.

Section snippets

Covalent coupling of capture Ab to FlowMetrix microspheres

Capture Abs (Table 1) were covalently coupled to FlowMetrix microspheres (beads) according to the manufacturer's instructions (Luminex). A mixture of water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) is used to activate free carboxyl groups on the beads. Then a covalent bond is formed between the activated carboxyl groups and free amino groups on the Ab. Sodium azide and any compound containing amino groups will interfere

Method development

Adapting the FlowMetrix system to quantify multiple cytokines was relatively straightforward. However, three issues required more attention. The first issue was maintaining the beads within the pre-set FlowMetrix gates, an important issue as these gates cannot be altered. Two factors were found to affect the FL2/FL3 stability of the beads; photobleaching and the level of FL1 fluorescence. We found that the beads were irreversibly photobleached by prolonged exposure to light causing them to fall

Discussion

The principal advantage of the FlowMetrix assay is that a large number of cytokines in a small sample can be quantified simultaneously. The same volume would only be sufficient for the analysis of a single cytokine by ELISA. Naturally, this advantage increases with each additional cytokine analyzed and can be translated into substantial savings in the cost of reagents and time required to perform the assay. The FlowMetrix assay may also facilitate a more thorough analysis of biological samples

Acknowledgements

We are grateful to Martin Dorf for recombinant TCA3. We also thank Marcia Blackman, Richard Cross and Dharmesh Desai for their critical review of the manuscript. This work was supported by the NIH (AI-39480), a Cancer Center Support CORE grant (5 P30 CA-21765) and the American Lebanese Syrian Associated Charities (ALSAC).

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