Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry, allergies vs. non-allergies
Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry, allergies vs. non-allergies
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DOI：
10.1016/s0022-1759(01)00407-0
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发表时间：
2001-08-01
影响因子：
2.2
通讯作者：
Barney, NP
中科院分区：
文献类型：
--
作者：
Cook, EB;Stahl, JL;Barney, NP
泪液通过提供对其众多功能至关重要的严格调节的最佳细胞外环境，在维持角膜和结膜完整性方面发挥重要作用，这些功能包括抗微生物防御，伤口愈合和炎症反应，如过敏。在各种眼部疾病状态的泪液中已经报道了升高水平的炎性细胞因子。泪液细胞因子的表征受到可获得的小体积（微升量）的限制。新开发的Becton Dickinson细胞微珠阵列（CBA）解决了这一局限性，它将“夹心”免疫测定的原理与流式细胞术的能力相结合，可同时测量多个颗粒的特征。该技术允许在单个泪液样品中同时测定六种人细胞因子（IFN γ、TNF α、IL-2、IL-4、IL-5、IL-10）浓度。从非过敏性（n = 7）和过敏性（n = 9）供体的下穹窿收集泪液。将每个泪液样品或细胞因子标准品与捕获Ab-珠试剂和检测Ab-藻红蛋白（PE）试剂的混合物一起孵育，并使用流式细胞术进行分析。所有六种细胞因子在非过敏性和过敏性泪液中都可检测到。来自过敏供体的泪液含有显著较少的IL-10（p = 0.035），并且TNF α/IFN γ的比率显著增加。IL-5/IFN γ和IL-5/IL-10（分别为p = 0.0008、0.0124和0.011）。流式细胞仪微珠阵列所需的小体积（5-10穆尔/测试）允许从单个泪液收集中测量所有六种细胞因子。这减少了收集时间，最大限度地减少了刺激对泪液中细胞因子浓度的混淆效应，并允许计算细胞因子比率。(C)出版社：Elsevier Science B. V.
Tears play an essential role in maintaining corneal and conjunctival integrity by providing a tightly regulated, optimal extracellular environment critical to its numerous functions, which include anti-microbial defense, wound healing and inflammatory responses such as allergies. Elevated levels of inflammatory cytokines have been reported in tears from various ocular disease states. Characterization of tear cytokines has been limited by the small volume (microliter amounts) attainable. This limitation was addressed with the newly developed Becton Dickinson Cytometric Bead Array (CBA), which combines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of multiple particles. This technique allows determination of six human cytokine (IFN gamma, TNF alpha, IL-2, IL-4, IL-5, IL-10) concentrations simultaneously in a single tear sample. Tears were collected from the inferior fornix of non-allergic (n = 7) and allergic (n = 9) donors. Each tear sample or cytokine standard was incubated with a mixture of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent, and analyzed using flow cytometry. All six cytokines were detectable in both non-allergic and allergic tears. Tears from allergic donors contained significantly less IL-10 (p = 0.035), and had significant increases in the ratios of TNF alpha /IFN gamma. IL-5/IFN gamma and IL-5/IL-10 (p = 0.0008, 0.0124 and 0.011, respectively). The small volume required (5-10 mul/test) by the Cytometric Bead Array allows measurement of all six cytokines from a single collection of tears. This decreases collection time, minimizing the confounding effect of stimulation on cytokine concentration in tears, as well as allowing calculation of cytokine ratios. (C) 2001 Published by Elsevier Science B.V.