Dionne Karen, Redfern Rachel L, Nichols Jason J, Nichols Kelly K
The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX.
Mol Vis. 2016 Feb 23;22:177-88. eCollection 2016.
Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups.
Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at -80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups.
Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1β, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray than the Luminex assay. Significant differences in expression levels (p<0.05) were only detected between the CL and DE groups for IL-7 and IL-8 and between the CL and NOR subjects for IL-8.
In addition to detecting more significant differences between the subject groups, the Quantibody® microarray detected more inflammatory cytokines in total within the range of detection than the Luminex assay. Differences were also noted in the types of cytokines each assay could detect from the limited protein samples. Both methods offer advantages and disadvantages; therefore, these factors should be considered when determining the appropriate assay for analyzing tear protein samples.
炎症介质已被证明可调节干眼（DE）疾病，且可能与疾病严重程度相关，但文献中使用的方法及相关研究结果差异显著。本研究的目的是比较定量微阵列和磁珠分析法这两种方法，用于检测三个受试者组提取的泪液样本中的细胞因子水平。
从20名软性隐形眼镜佩戴者（CL）、20名正常非隐形眼镜佩戴者（NOR）和20名DE受试者的左右眼Schirmer试纸条收集泪液，并储存在-80°C。使用碳酸氢铵和丙酮洗脱并沉淀泪液蛋白。将每个受试者的左右眼样本合并。按照Bradford蛋白定量法，取10μg总蛋白用于两种分析，即Quantibody®人炎症阵列3（RayBiotech）和高灵敏度人细胞因子磁珠试剂盒（Millipore）。分别使用GenePix® 4000B扫描仪（Molecular Devices）或Luminex MagPix®酶标仪（Luminex）进行检测。然后比较两种仪器和三个受试者组之间的数据。
在Quantibody®微阵列上的40种蛋白质中，有7种的平均表达水平高于检测下限：细胞间黏附分子-1（ICAM-1）、单核细胞趋化蛋白-1（MCP-1）、γ干扰素诱导单核因子（MIG）、巨噬细胞集落刺激因子（MCSF）、金属蛋白酶组织抑制因子-1（TIMP-1）、金属蛋白酶组织抑制因子-2（TIMP-2）和肿瘤坏死因子受体I型（TNF-RI）。使用Student t检验时，CL组和DE组之间在MCSF、TIMP-1和TNF R1的表达水平上存在显著差异（p<0.05），NOR组和DE组之间在ICAM-1的表达水平上存在显著差异，CL组和NOR组之间在ICAM-1、MCP-1、MCSF、TIMP-1、TIMP-2和TNF-R1的表达水平上存在显著差异。在用Luminex检测的13种蛋白质中，白细胞介素-1β（IL-1β）、白细胞介素-4（IL-4）、白细胞介素-6（IL-6）、白细胞介素-7（IL-7）和白细胞介素-8（IL-8）的表达水平高于最低可检测水平，与Quantibody®微阵列相比，这些蛋白质最常通过Luminex检测到。相反地，白细胞介素-2（IL-2）、白细胞介素-12（IL-12）、白细胞介素-13（IL-13）、γ干扰素（INF-γ）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）通过Quantibody®微阵列检测到的频率高于Luminex检测。仅在CL组和DE组之间的IL-7和IL-8以及CL组和NOR受试者之间的IL-8的表达水平上检测到显著差异（p<0.05）。
除了检测受试者组之间更显著的差异外，Quantibody®微阵列在检测范围内总共检测到比Luminex检测更多的炎症细胞因子。在从有限的蛋白质样本中每种检测方法能够检测到的细胞因子类型上也存在差异。两种方法都有优点和缺点；因此，在确定分析泪液蛋白质样本的合适检测方法时应考虑这些因素。
