Antibody protein array analysis of the tear film cytokines.
Antibody protein array analysis of the tear film cytokines.
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DOI：
10.1097/opx.0b013e3181824e20
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发表时间：
2008-08
期刊：
影响因子：
--
通讯作者：
McNamara NA
中科院分区：
文献类型：
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作者：
Li S;Sack R;Vijmasi T;Sathe S;Beaton A;Quigley D;Gallup M;McNamara NA
据报道，包括细胞因子在内的许多生物活性蛋白会增加干眼病的发生，尽管炎症介质的具体特征和浓度因研究而异。这种变异性部分是由于使用灵敏度相对较低的斑点 ELISA 方法在有限的样品体积中定量低丰度蛋白质的固有困难造成的。使用通过各种技术收集的混合样本以及个体泪液蛋白昼夜模式的内在变化带来了额外的复杂性。目前的研究描述了蛋白质组学领域的最新进展，该进展使得能够鉴定人类泪液样本中的数十种低丰度蛋白质。市售的固定相抗体蛋白阵列经过改造，提高了小体积生物液体分析的适用性，特别强调泪膜蛋白质组学。阵列经过改造，可以同时筛选从个体眼睛收集的少量泪液样本中的一组炎症细胞因子。进行了一项初步研究，比较了一小部分干燥综合征患者的泪液阵列结果。由于泪液的独特性质，泪液中细胞因子的多重微板阵列测定提出了意想不到的挑战。对塑料具有亲和力、捕获抗体和 IgG 并产生一系列复杂的基质效应的因素的存在，深刻影响了点 ELISA 的可靠性，包括背景反应性水平升高和结合目标蛋白的能力降低。使用从干燥综合征患者身上收集的眼泪进行的初步结果揭示了蛋白质阵列技术的方法学优势，并支持自身免疫介导的干眼病具有炎症成分的概念。他们还强调了在解释微孔阵列结果时可能面临的固有困难，这些困难是由光晕效应、基质效应、图像饱和以及捕获抗体和探针抗体之间的串扰造成的，这些困难会大大降低信噪比并限制获得有意义结果的能力。
Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples. Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes. A preliminary study comparing tear array results in a small population of Sjögren’s syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein. Preliminary results using tears collected from patients with Sjögren’s syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.