Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay
Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay
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DOI：
10.1016/s0022-1759(99)00069-1
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发表时间：
1999-07-30
影响因子：
2.2
通讯作者：
Vignali, DAA
中科院分区：
文献类型：
--
作者：
Carson, RT;Vignali, DAA
生物液和组织培养样品中细胞因子和趋化因子的定量方法包括生物测定法、酶联免疫吸附试验(ELISA)、细胞内染色、核糖核酸酶保护试验(RPA)和聚合酶链式反应(PCR)。然而，这些技术中的每一种都有一个或多个显著的局限性。在这里，我们描述了一种使用FlowMetrix(TM)系统的新的多重分析，它可以在小样本体积内同时定量多个细胞因子。该方法比常规微量酶联免疫吸附试验更准确、更灵敏、更具重复性。此外，所涉及的时间和成本与ELISA法相当或更少。FlowMetrix(TM)分析的一个关键特征是它的多重能力：在这里，我们表明该方法可以准确地定量100亩L样本中的15种细胞因子，而用ELISA进行相同的分析需要1.5ml(每个细胞因子分析100亩L)。通过使用这种Flow Metrix(TM)分析，我们可以证明只有T辅助1(T(H)1)变异的细胞产生可检测到的水平的IL-2，而只有T(H)2变异的细胞产生显著数量的IL-4。两个T细胞亚群都产生了其他六种细胞因子，其中T(H)1群体产生更多的IL-3、粒细胞-单核细胞集落刺激因子(GM-CSF)和干扰素(干扰素)-γ，T(H)2群体产生更多的IL-5、IL-10和IL-13。其他七种细胞因子的产生没有检测到数量。这个测试应该被证明是一个强有力的工具来定量细胞因子，或任何其他可获得抗体对的可溶性产品。它还将提供免疫反应过程中分泌过多细胞因子的更完整的图景。(C)1999 Elsevier Science B.V.保留所有权利。
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(TM) 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(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 same analysis by ELISA requires 1.5 ml (100 mu l for each cytokine assay). By 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 other cytokines were produced by both T cell subsets, with the T(H)1 population 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 available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response. (C) 1999 Elsevier Science B.V. All rights reserved.