基于磁珠复合法的撕裂细胞因子分析
Summary
泪膜细胞因子的分析有助于研究各种眼部疾病。基于微珠的复合检测方法简单、灵敏, 能够对小体积样品中的多个目标进行测试。在这里, 我们描述了一个协议的撕裂膜细胞因子分析一个使用珠基复用法。
Abstract
泪膜是一种复杂的脂质、蛋白质和矿物质的混合物, 覆盖了眼睛的外表面, 从而为底层细胞提供润滑、营养和保护。泪流分析是鉴别各种眼部疾病的预测、诊断和预后的标志物的一个新兴领域。眼泪是容易接近, 他们的收集是无创的。因此, 在研究蛋白质或代谢物组成的变化及其与病理条件的关系时, 先进的技术正日益突出, 以识别多分析的泪水。泪液细胞因子是研究眼部健康状况的理想标志物, 也有助于了解眼部疾病和春季结膜炎等不同眼表疾病的发病机制。基于微珠的复用检测具有较高的灵敏度, 能在少量样品中探测多个分析。在这里, 我们描述了一个标准的催泪样本收集, 提取和分析细胞因子的特征, 采用微珠的多重检测。
Introduction
眼泪是由泪腺和副腺体产生的, 并覆盖眼睛的外表面。泪膜由外脂层和内水层组成, 包括可溶性蛋白、粘和膜结合粘。眼泪可以防止微生物侵入, 提供营养, 并为眼部表面润滑。眼泪作为空气和组织之间的接口, 用于氧气输送到角膜。1撕裂膜由蛋白质、碳水化合物、血脂和电解质组成。一些研究发现, 泪液蛋白与眼部和全身性疾病如青光眼、干眼病、春季结膜炎、糖尿病、甲状腺相关眼眶和癌症之间的关联。2,3,4撕裂样品可以通过 microcapillary 管或撕裂流条 (斯戈默条) 收集。此外, 斯戈默的测试是在角膜和屈光手术诊所中执行的标准程序, 其结果可用于细胞因子分析化验。无创样本采集, 生物标本的可及性, 撕裂成分与各种生理和病理条件的联系, 使泪膜成为几种眼部和全身性疾病的生物标志物的潜在来源。4,5,6
泪液细胞因子在研究各种眼病的眼表健康和炎症状态方面起着重要的作用。7撕裂标本中几种细胞因子的异常浓度报告与干眼病、春季结膜炎 (VKC)、特应性结膜炎 (AKC)、季节性过敏性结膜炎和葡萄膜炎有关。8,9,10,11,12,13撕裂蛋白可以用质谱、印迹和酶联免疫吸附法等传统方法进行分析。14,15然而, 这些方法的局限性是敏感性差和大量的样本需要分析的多个撕裂细胞因子在每个病人。16,以17微珠为基础的复配分析方法, 对复杂混合样品中的多分析进行了研究, 成功地应用于撕裂样品, 分析了不同疾病中的多种细胞因子。6,18结合了三明治 elisa 和流式细胞术技术, 使得这些检测方法比 elisa 更灵敏, 可以对单个样本中的多个分析进行量化。19该方法可应用于多种临床标本和细胞培养清, 有助于研究几种病理生理条件下的免疫应答。20,21,22,23,24
有几项研究比较了珠基多重检测与 ELISA 法的相关性, 并报告了这些方法之间的相互关系。厕所et al.比较了基于微珠的多重检测与常规 ELISA 法测定人血清或血浆样品中脂联素、抵抗素、瘦蛋白和生长激素的含量, 并报告了两者之间的强相关性 (r 和 #62; 0.9)。25杜邦et al.报告了在 IL-6 和脂多糖刺激全血的 IL-1β、IL-4、IL-5、IL-10、节能、干扰素-植物和 TNF-α的检测中, 珠基复合测定与 ELISA 之间的强相关性。从怀孕妇女收集。26平克林et al.报告了另一种强关联度检测血清抗体对乙型流感嗜血杆菌(r = 0.96), 毒素的测定破伤风梭菌 (r = 0.96) 和杆菌白喉(r = 0.91)。27 Biagini et al.报告了一个高正相关 (r = 0.852) 之间的珠基复合测定和 ELISA 检测的芽孢杆菌抗体在血清样品中的抗 PA IgG。28 Wang et al.报告了在检测阿尔茨海默病生物标志物淀粉样蛋白β 42 (r = 0.77), 总 tau (r = 0.94), 和头磷酸化的氨基酸 181 (r = 0.82) 之间的相关性。脑脊液样本29这些研究证明了基于微珠的多重分析方法对临床样品的多样性、小样本量的要求以及与标准 ELISA 的相关性, 使微珠基复合检测成为一个有前途替代传统的 ELISA 方法检测不同类型的样品中的多种分析的疾病表型。在这里, 我们描述了一个标准化的协议, 以珠子为基础的多重检测细胞因子分析41分析的撕裂样本从健康的主题使用斯戈默带。
Protocol
本研究中使用的协议已获新加坡谭家医院机构审查委员会批准. 1. 撕裂细胞因子分析 催泪集: 要求受试者舒适地坐在检查椅上, 并将其头部置于头枕上. 请主题查找, 仔细打开斯戈默条 (由惠特曼滤纸41制成), 并将条的圆端放在眼睛的下穹上, 并指示主体闭上眼睛5分钟。在收集眼泪时, 要非常小心, 尽量减少眼睛表面接触。 …
Representative Results
用泪流条和细胞因子水平分析了8眼4健康受试者的泪液样本。所有科目均为男性, 四受试者的年龄分别为36、42、44及52岁。通过临床试验 (泪膜破裂时间、斯戈默试验、角膜染色、结膜染色、眼睑/板腺检查、角膜撕裂征象和视觉体征及症状), 对眼部健康和干眼病进行评估。国际干眼研讨会, 2007 指导方针, 没有一个主题显示任何迹象和症状的干眼病。31 <p …
Discussion
细胞因子是小细胞分泌的蛋白质和有效的免疫调节调控免疫应答。32撕裂标本中各种细胞因子的表达谱与眼睛的各种病理条件有关, 研究细胞因子图谱有助于了解疾病发病机制, 确定眼部健康状况,疾病的严重性, 诊断和进展。2,5较低的蛋白质浓度和小样本量是传统方法的主要挑战, 限制了对撕裂细胞因子的分析, 如 ELISA 法的使用。基于<su…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项研究工作得到由谭医院个人种子资助计划2015中心资助;新加坡眼科研究所的试点补助金和谭医院的资金补助。
Materials
| Milliplex MAP human cytokine / chemokine magnetic bead panel -1 kit | Merck, USA | HCYTOMAG-60K-41 | |
| Flexmap 3D luminex instrument | Luminex Corp, Austin, TX, USA | ||
| xPonent software | Luminex Corp, Austin, TX, USA | ||
| RBXGenerator software | BIO-RAD, France | ||
| Bio-Plex Manager 6.1 | BIO-RAD, France | ||
| Plate shaker | Corning, USA | ||
| TECAN Microplate Washer | Tecan, Switzerland | HydroSpeed | |
| Vortex Genie 2 | Scientific Industries inc, USA | G560E | |
| Pipettes | Mettler Toledo, CA, USA | ||
| 1.5ml microcentrifuge tube | Axygen, USA | MCT-150-C | |
| Schirmer tear flow test strip | Eye Care and Cure, USA | 101657 | |
| Flexmap 3D Calibration Kit | Luminex Corp, Austin, TX, USA | 40-028 | |
| Flexmap 3D Verfication Kit | Luminex Corp, Austin, TX, USA | 40-029 | |
| Ocular examination chair |
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Balne, P. K., AU, V. B., Tong, L., Ghosh, A., Agrawal, M., Connolly, J., Agrawal, R. Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles. J. Vis. Exp. (128), e55993, doi:10.3791/55993 (2017).