印制糖类阵列是分析小动物循环抗碳水化合物抗体汇辑的一种敏感技术
1Infectious Pathology and Transplantation Division,Bellvitge Biomedical Research Institute (IDIBELL), 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (IBCh),Russian Academy of Sciences, 3Semiotik LLC, 4Auckland University of Technology, 5Intensive Care Department,Bellvitge University Hospital
Summary
这项工作显示了印刷糖类阵列 (pga) 技术在小动物循环抗碳水化合物抗体分析方面的潜力。
Abstract
特定个体的循环抗碳水化合物抗体的表达物往往与其免疫学状态有关。不仅个人免疫状况决定了对抗内部和外部潜在威胁信号的成功, 而且还决定了特定的循环抗糖类抗体模式的存在 (及其血清学水平的变化) 可能是一种某些病理条件的发病和进展的重要标志。在这里, 我们描述了一种基于印刷的字形阵列 (pga) 方法, 它提供了一个以非常高的灵敏度测量数百个乙二醇目标的机会;使用最小数量的样本, 这是一个常见的限制, 当小动物 (老鼠, 老鼠, 仓鼠等) 被用作模型, 以解决人类疾病的各个方面。作为这种方法的一个有代表性的例子, 我们展示了从分析 balb/小鼠天然抗糖类抗体的数量中获得的结果。我们证明, 每个参与研究的 balb1 小鼠, 尽管在基因相同和在相同条件下保持, 发展了一个特定的模式的天然抗碳水化合物抗体。这项工作声称扩大使用 pga 技术, 以调查曲目 (特殊性) 和循环抗碳水化合物抗体的水平, 无论是在健康和在任何病理条件。
Introduction
抗体通过激活补体系统 4,5, 直接中和病毒1、2和细菌2、3, 在我们抵御入侵病原体方面发挥着核心作用和促进吞噬6。此外, 它们是癌症靶向和消除恶性细胞7和稳态维持的基本要素.
免疫系统的疾病会导致自身免疫性和炎症性疾病10 和癌症11。所有这些病理条件理想地要求迅速诊断为有效的治疗。在自身免疫性疾病的情况下, 血清学存在的自身抗体在大多数情况下是一个预测的诊断自身免疫 10,12。这些抗体与细胞表面和细胞外自身抗原发生反应, 它们往往存在多年之前的自身免疫性疾病10,12。免疫缺陷和癌症也被诊断为血液检测, 要么测量抗体等免疫元素的水平, 要么测量其功能活动 11。
确定循环抗体的曲目及其血清学水平是最重要的, 以建立一个预后, 并评估所有上述病理条件的进展。我们之前已经证明了 pga 技术在分析不同动物物种中的循环抗体的潜力13-16, 最大限度地减少了大量血清学样本的使用, 避免了这一问题与抗体交叉反应17相关, 并允许对大量抗体进行高通量分析 15.
甘氨酸免疫检测主要是由碳水化合物的来源和产生决定的因素, 其中决定了配体15,18,19, 20的亲和力和结合,21。基于甘胶的免疫检测可以在悬浮液 (微球)15,21,22 或平激活表面15,21,22, 23,24。最后一种包括 elisa (其中最传统的方法) 和 pga。在同一实验环境中, 没有多少数据可以比较这些方法, 即15、25、26、27。我们之前比较过这些免疫检测的有效性和选择性来分析单个人血浆样本中的抗糖类抗体15。对于一些抗体, 如针对抗阿苏血组的抗体, 所有的免疫检测都能检测到它们的统计意义, 它们彼此呈正相关。同时, 抗 p1 抗体主要由具有最高判别力的 pga 检测, 不同的糖类免疫检测 15,18 的测定没有相关性,21. 这些方法之间的差异主要与抗体抗原比和糖类取向15有关。elisa 和悬浮液阵列比 pga 更容易受到非特异性结合, 因为在这些方法中, 抗原超过抗体15。此外, 在 pga 中, 糖类的取向比 elisa 和悬浮阵列15中的方向受到更多的限制。elisa 是方便的, 当研究包括一个有限的面板的糖类。除了悬浮液阵列外, elisa 还在分析重构方面提供了更广泛的灵活性。pga 是特别方便的发现方法15,18,21,28。尽管有这些明显的优点和缺点, 但上述三种免疫检测方法可以用来研究糖类抗体相互作用的不同方面。这项研究的最终目标是指导选择更合适的方法。
本工作旨在扩大 pga 技术在小动物循环抗糖类抗体检测中的应用。作为一个有代表性的结果, 我们在这里提出了一个详细的协议, 以评估体内天然抗碳水化合物抗体的反应在成年 balb1 小鼠 pga。
Protocol
1. 糖片生产 微阵列制备 用非接触式机器人排列器 (落模量 ~ 900 pl), 在6个复制的 n-羟基琥珀衍生玻璃滑梯上, 以 300 mm 磷酸盐缓冲盐水 (pL, ph 8.5) 将糖类 (50 mm) 和多糖 (10μg/ml) 打印成铝。每张幻灯片包含4个不同的子数组块 (图 1a, 颜色) 重复6次。每个子数组由1B 不同的糖原点组成, 包括控件 (8 行 x14 列) (图 1b)。注: 与圣枪有?…
Representative Results
在这里, 我们提出了一个代表性的结果, 从定量的天然抗糖类抗体的曲目在 20个 balb1 小鼠的人群中获得。本研究中使用的糖基含有419种不同的糖类结构。大多数糖类被合成为-ch2 ch 2 ch2 nh2间隔武装o-糖苷, 在几个情况下作为-ch2 nh 2 或 -nhch 2 nh 2 苷. 所有的糖类结构都用高分辨率 (700-或 800 mhz) 核?…
Discussion
乙二醇微阵列已成为研究蛋白质-糖相互作用的不可缺少的工具.本文介绍了一种基于 pga 技术的研究 balb1 小鼠抗碳水化合物抗体循环的协议。由于 pga 提供了筛选大量生物未知糖类的可能性, 因此它是一种非常方便的发现工具13、15、28。该方法提供了在相同的实验环境中使用减少的血清学样本 (50μl) 测量数百种…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了西班牙卫生部 carlos iii 卫生研究所的 PI13/01098 赠款 “卫生调查基金会” 的支持。db-g 受益于欧洲联盟第七个框架方案 (fp7/2007-2013) 根据《赠款协定》 603049 (translink) 资助的博士后研究职位。nk、ns 和 nb 的工作得到了俄罗斯科学基金会 #14 50-00131 赠款的支持。db-g 希望感谢 marta broto、j. pablo salvador 和 ana sanchis 在统计分析方面提供的出色技术援助, 并感谢亚历山大·拉基特科提供的协助。在 “加泰罗尼亚总司工业大学” 的支持下 (赠款编号 2018年 di 021)。我们感谢 cerca 方案/加泰罗尼亚总署提供的机构支助。
Materials
| Antibodies | |||
| biotinylated goat anti-human Igs | Thermo Fisher Scientific, Waltham, MA, USA | Ref. #: 31782 | |
| biotinylated goat anti-mouse IgM + IgG | Thermo Fisher Scientific | Ref. #: 31807 | |
| Equipment | |||
| Robotic Arrayer sciFLEXARRAYER S5 | Scienion AG, Berlin, Germany | http://www.scienion.com/products/sciflexarrayer/ | |
| Stain Tray (slide incubation chamber) | Simport, Beloeil, QC, Canada | Ref. #: M920-2 | |
| Centrifuge | Eppendorf, Hamburg, Germany | Ref. #: 5810 R | |
| Pipettes | Gilson, Middleton, WI, USA | http://www.gilson.com/en/Pipette/ | |
| Slide Scanner | PerkinElmer, Waltham, MA, USA | ScanArray GX Plus | |
| Shaking incubator | Cole-Parmer, Staffordshire, UK | Ref. #: SI50 | |
| Biological samples | |||
| BALB/c mice sera | This paper | N/ A | |
| Complex Immunoglobulin Preparation (CIP) | Immuno-Gem, Moscow, Russia | http://www.biomedservice.ru/price/goods/1/17531 | |
| Chemicals, Reagents and Glycans | |||
| Glycan library | Institute of Bioorganic Chemistry (IBCh), Moscow, Russia | N/ A | |
| Bovine serum albumin (BSA) | Sigma-Aldrich, St. Louis, MO, | Ref. #: A9418 | |
| Ethanolamine | Sigma-Aldrich | Ref. #: 411000 | |
| Tween-20 | Merck Chemicals & Life Science S.A., Madrid, Spain | Ref. #: 655204 | |
| Phospahte buffered saline (PBS) | VWR International Eurolab S.L, Barcelona, Spain | Ref. #: E404 | |
| Sodium azide | Sigma-Aldrich | Ref. #: S2002 | |
| Streptavidin Alexa Fluor 555 conjugate | Thermo Fisher Scientific | Ref. #: S21381 | |
| Streptavidin Cy5 conjugate | GE Healthcare, Little Chalfont, Buckinghamshire, UK | Ref. #: PA45001 | |
| Materials | |||
| N-hydroxysuccinimide-derivatized glass slides H | Schott-Nexterion, Jena, Germany | Ref. #: 1070936 | |
| Whatman filter paper | Sigma-Aldrich | Ref. #: WHA10347509 | |
| 1.5 mL tubes | Eppendorf | Ref. #: 0030120086 | |
| Software and algorithms | |||
| ScanArray Express Microarray Analysis System | PerkinElmer | http://www.per | |
| kinelmer.com/microarray | |||
| Hierarchical Clustering Explorer application | University of Maryland, MD, USA | http://www.cs.umd.edu/hcil/hce/ | |
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Cite This Article
Olivera-Ardid, S., Khasbiullina, N., Nokel, A., Formanovsky, A., Popova, I., Tyrtysh, T., Kunetskiy, R., Shilova, N., Bovin, N., Bello-Gil, D., Mañez, R. Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals. J. Vis. Exp. (144), e57662, doi:10.3791/57662 (2019).