Name: simultaneous distinction of monospecific and mixed dfs70 patterns during ana screening with a novel hep-2 elite/dfs70 knockout substrate
Uploaded: 2018-01-17T10:50:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate at JoVE.com

我们感谢安德鲁曾格执行 IIF 实验和收集图像。

该议定书遵循三一生物技术机构人类研究伦理委员会的指导方针。
1. 样品和基质制备
<ol><li>允许包含衬底幻灯片的邮袋 (每张幻灯片上有12口井, 包含 HEp2 或 HEp-2+HEp2 高细胞混合物), 以平衡到室温, 以获得最佳性能, 防止在滑动面上凝结湿气。样品的加法。这大概采取在 10-15 min 之间。</li>
	<li>一旦平衡到室温, 小心地用手指移除基底片, 而不触及袋的两侧。</li>
	<li>标签的幻灯片, 并放置在一个孵化室 (室温) 内衬纸毛巾蘸水, 以防止干燥的幻灯片样品和共轭孵化。 注: 干井可产生非特异性染色;在孵化室的湿纸巾提供湿度和防止良好的干燥。</li>
</ol>2. 添加控制和样品
<ol><li>免除大约50µL 的阴性/阳性对照, DFS70-ANA 阳性血清的组合, 如本研究所述, 或患者血清样本到个别滑动井。</li>
	<li>将控制和样本放入幻灯片中的每个井中。</li>
	<li>为了防止污染, 避免溢井。在不溢井的情况下, 确保井面覆盖完整, 避免气泡。 注意: 建议的卷是50µL, 按步骤2.1。</li>
	<li>将盖子放在孵化室上, 在室温下孵育30分钟的幻灯片。如果病人的血清中有任何的 ANA, 它将会在这个潜伏期内与每一个井中的细胞结合。</li>
	<li>一旦孵化期结束, 从孵化室中取出滑梯。保持在 tab 端的幻灯片和温和冲洗约10毫升磷酸缓冲盐 (pbs) 洗涤缓冲区提供的试剂盒, 使用吸管或在一个烧杯填充 pbs 10-十五年代. 将幻灯片立即转换为 Coplin jar, 并使用 pbs 洗涤缓冲液 (浸泡) 10 分钟, 对所有剩余的幻灯片重复该过程。 注意: 当在 Coplin jar 中放置多个幻灯片时, 请确保幻灯片的单元格侧不与另一张幻灯片接触, 因为这将导致损坏玻璃幻灯片上的单元格。</li>
</ol>3. 添加荧光共轭
<ol><li>一次只从 Coplin jar 中移除一张幻灯片。要从幻灯片上删除多余的 PBS 洗涤缓冲区, 请轻轻地轻拍或涂抹纸巾上的幻灯片。每个试剂盒上都配有 FITC 标记的人 IgG 荧光共轭。对每一个井, 轻轻地应用1滴 (约50µL) 的所提供的共轭。</li>
	<li>在闭合的染色孵化室中孵育幻灯片30分钟。 注意: 建议使用 Fc 特异性 IgG 共轭。如果患者的血清中有任何的 ANA, 共轭物将与在这个潜伏期的每个井中存在的细胞结合, 这会导致在井中的细胞的特定荧光。共轭物对光敏感。封闭的孵化室将有助于防止光干扰的共轭反应的细胞存在于每个井。</li>
	<li>一旦孵化期结束, 从孵化室中取出滑梯。保持在标签端的幻灯片和温和冲洗与约10毫升 pbs 洗涤缓冲区提供的套件, 使用吸管或烧杯填充 pbs。将滑块立即转到带有 PBS 洗涤缓冲液的 Coplin 罐中, 并洗涤 (浸泡) 10 分钟. 对所有剩余的幻灯片重复该过程。 注意: 当在 Coplin jar 中放置多个幻灯片时, 请确保幻灯片的单元格侧不与另一张幻灯片接触, 因为这将导致损坏玻璃幻灯片上的单元格。</li>
	<li>将所提供的片放在干纸巾上。安装介质随套件一起提供。到片的底部边缘, 在一条线上应用 3-4 滴安装介质。</li>
	<li>从包含洗涤缓冲器的 Coplin 缸中一次取出一张幻灯片。要去除多余的洗涤缓冲器, 轻轻拍打或涂抹纸巾上的滑块。</li>
	<li>通过将幻灯片的下边缘置于片的边缘, 将幻灯片轻轻地放在片上。这使得在下边缘片上的安装介质可以流向幻灯片的上边缘, 并最大限度地减少气泡的形成, 这可能会干扰幻灯片的每个井的读数。 注: 应使用最小压力将覆盖幻灯片安装到幻灯片上。任何过量的压力都会导致盖板的横向移动。这会对细胞的单层制备造成损害, 并导致畸变。</li>
	<li>将幻灯片存储在 2-8 ° c 的黑暗中。在安装后或48小时内检查幻灯片。 注: 较长的存储可能导致模式和荧光信号的恶化。</li>
</ol>4. 确定正反结果
<ol><li>使用 FITC 过滤器设置的荧光显微镜查看幻灯片, 位于暗室中。</li>
	<li>在荧光显微镜下, 在200X 或更大的放大倍数下检测特定的亮绿色荧光, 以使其对阳性和模式进行最终解释。观察积极和消极的控制。 注: 阳性对照将在细胞核内显示明亮的绿色荧光 (图 2A, 均质)。在荧光显微镜下, 阴性对照将不显示特定的绿色荧光。</li>
	<li>记录每个井的正/负结果, 并包括阳性病例的 ANA 模式。</li>
</ol>

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作者· Malyavantham 和拉克什曼南是美国三一生物技术公司的员工。

细胞核和细胞质抗原的自身抗体在系统性自身免疫性疾病中非常普遍。虽然没有单一的化验提供最好的灵敏度和特异性, IIF 被广泛认为是一种高度敏感和 cost-effective 的系统性自身免疫疾病的筛选方法2,3,4.尽管 iif 协议的盛行已超过50年, 但 iif 测试的准确性非常依赖于技术人员的技能, 仔细执行协议的各个步骤, 并且使用维护良好的荧光显微镜。系统性自身免疫性疾病的诊断不应基于单一血清学测试的结果, 如 IIF 的 HEp-2。
使用优质水 (蒸馏/去离子), 使用标准化的试剂盒组件 (HEp-2 细胞单分子膜, 样品稀释剂, 正负控制, pre-diluted 优化 FITC 共轭, 安装介质)对结果有积极的影响。在井中跳过添加控件或样本会助长虚假的负面解释。在添加样品或控制后, 在任何点干燥的幻灯片会导致 artifactual 假阳性反应和/或不正确的模式。在安装介质分配前, 滑动或干燥滑动井的洗涤缓冲器过多会导致工件的产生。在放置片之前, 在滑动面上添加足够数量的安装介质或生成气泡会导致在显微镜下观察时出现模糊或对焦不清晰的反应。安装的幻灯片必须存储在 2-8 ° c, 并在建议的时间窗口分析, 以尽量减少假阴性或 artifactual 结果。
使用一个保养良好的 epi 荧光显微镜, 它有适当的 LED/汞/氙光源和清洁的光学元件, 包括未损坏的励磁和发射过滤器, 对于获得准确的 IIF 结果至关重要。最终用户对辨别正/负反应强度和模式解释的培训是至关重要的。HEp-2 IIF 的检测是易受缺乏标准化的程序, 显微镜配置, end-user 训练或技能。在教育目的4中, 以各种制造商获得的一致的命名和代表性的模式和图像的例子, 都是由汇联 (< www.ANApatterns.org >) 提供的。HEp-2 细胞模式分类树提供的各种核, 细胞质和有丝分裂模式是一个宝贵的资源, 以了解不同的模式之间的微妙差异, 可能看起来类似于未经训练的眼睛4。一个具有挑战性的模式的主要例子是 DFS70, 也缺乏一个明确的联系与自身免疫状态, 并在前面的章节讨论这种模式是非常普遍的健康人群5。
根据研究, anti-DFS70 自身抗体的患病率在健康人群、ana 筛查群体和无系统性自身免疫疾病证据的 ana 阳性个体之间有差异9,16,20 ,30,31。据报道, DFS70 自身抗体的发生与其他与疾病相关的安娜模式的分离。在 2-22% 的健康对照报告中, 孤立或 monospecific DFS70 模式, 但在不到1% 的情况下与自身免疫性风湿性疾病32。基于这些趋势, 提出了 DFS70 单阳性模式作为排除自身免疫性风湿性疾病的标准, 其方法为:3,31,32。为促进自身抗体测试命名和 HEp-2 IIF 结果的解释的统一而设立的联汇会委员会建议在报告 ANA 筛选结果4时报告 DFS70 阳性。
在排除基于 DFS70 单阳性的系统性自身免疫性疾病之前, 必须对目前的筛查和确认方法 (特别是 DFS70) 的现状进行回顾。DFS70 阳性的协议 HEp-2 IIF 和固相验证性检测, 即 ELISA, 评估, 和线免疫分析/斑点杂交方法) 之间的差异广泛不同的研究13,22,25,33.对这一分歧作出贡献的 IIF 解释和验证性化验参数已经讨论过了5,13,34。最近提出的 anti-DFS70 抗体确认的免疫方法解决了当前固相检测的一些缺陷 (对于 DFS70), 但要求实验室在 IIF 过程中对疑似样本执行额外的步骤, 这增加报告结果13的成本和周转时间。当 HEp-2 ELITE/DFS70 高细胞用于常规的 ANA 筛查时, 不需要额外的步骤。这降低了总体成本, 而且在初始筛选步骤27中可看出 DFS70 模式时, 对周转时间没有影响。使用传统的 HEp-2 IIF 方法的另一个缺点是在筛选阶段无法区分 DFS70 模式 tfbs 与其他 ANA 模式。然而, 这种缺点是克服了使用 HEp-2 ELITE/DFS70 高细胞27。
常规 HEp-2 和新工程 HEp-2 (HEp-2 ELITE/DFS70 KO) 的比较评价使用 ANA 阳性控制及其与单阳性 DFS70 控制的组合, 证明了新基板的功能同时用于阿纳斯和确认 DFS70 模式的屏幕 (图 1)。新基板的 iif 协议与传统的 HEp-2 iif 方法相同。所有与疾病相关的 ANA 模式的解释方案与 DFS70 模式的例外情况相同 (表 1)。使用新方法时, 对单阳性和混合 DFS70 模式的解释相对比较容易, 而且不需要额外的化验 (图 1B)。这一新方法在临床实验室的实施简化了与 DFS70 模式的解释相关的挑战, 并通过进一步揭示以前隐藏的与疾病相关的 ana 模式, 提高了 ana 筛查的整体准确性。DFS70 (混合模式)。

阿纳斯是自体免疫介导的系统性结缔组织病的一个标志1。采用几种方法对阿纳斯进行筛选和确认。使用 HEp-2 基板的 IIF 技术仍然是筛选阿纳斯12的最广泛使用和常用的推荐方法。尽管在固相诊断分析方法, 如酶联免疫吸附试验 (ELISA), 酶免疫分析 (EIA), 化学发光免疫分析 (评估), 和 bead-based 多重分析的进展, IIF 的 HEp-2 是最普遍的筛选方法由于其诊断有用性和成本效益1,2,3。above-mentioned 检测技术依赖于一组有限的纯化本机或重组抗原, 而在玻璃滑动井上的 HEp-2 细胞单层制剂呈现出大量抗原的自然形式, 分布在细胞核和细胞质, 与患者血清或阳性对照的自身抗体发生反应, 产生多种与自身免疫条件相关的模式。这些模式分为核、细胞质和有丝分裂模式的基础上的抗原在细胞中的位置。每个模式和相关的抗原/抗原和疾病状态都有很好的特征4。在 IIF 方法中, 患者和控制血清在玻璃滑动井中孵化, 这种 HEp-2 细胞的单层培养适当固定, 以保护其自然构型中的许多抗原。患者血清或阳性对照中的自身抗体可以与基底上 HEp-2 细胞 (玻璃滑动井) 在孵化过程中所呈现的抗原结合。后潜伏期, 未绑定的抗体被冲走, igg 类抗体检测荧光/荧光素异硫氰酸酯 (FITC) 共轭人 igg 试剂。未绑定报告-共轭被冲走, 玻璃片安装在幻灯片的顶部使用安装介质, 保存的反应和促进观察下的荧光显微镜。在装有合适的激发发射过滤器组合的荧光显微镜下观察反应 (对于 FITC 记者来说, 诊断显微镜通常使用的过滤器与励磁范围为467-498 毫微米和发射范围 513-556 毫微米)。安娜的存在是由一个明亮的绿色荧光的特定结构在细胞中表现出来的。与自动化的测试平台不同, IIF 方法依赖于连续稀释血清的费力过程和需要大量用户专业技能5,6的染色模式的可视化阳性检测。尽管有这些限制, HEp-2 仍然是最广泛使用和推荐的方法来筛选的阿纳斯由于标准化努力, 以模式解释和命名标准的国际共识的全日空模式 (汇联) 委员会,增强了 IIF 幻灯片处理的自动化解决方案的可用性, 以及结果解释3。
在 HEp-2 IIF 方法检测到的众多模式中, 针对psip1基因产品的自身抗体 (也称为 LEDGF/p75/DFS70), 近年来由于几个原因引起了极大的兴趣4,5 ,6,7,8,9,10。DFS70 抗体存在于高滴在健康的控制, 到目前为止的研究没有表现出一个独特的临床协会的存在 DFS70 自身抗体7,8,9 ,10,11,12,13。不同疾病状态和健康人群中 DFS70 抗体阳性结果的报告率差异很大, 研究6,8,9,10,14 ,15,16,171819202122 ,24,25,26,27,28。monospecific DFS70 模式是很好的区别, 从一个同类或斑点模式, 但解释混合均匀斑点图案和 anti-DFS70 抗体 tfbs 与其他阿纳斯是挑战, 甚至对专家读者。当前世代的 IIF 筛选方法和 DFS70 的具体固相检测不能区分 monospecific DFS70 反应从混合模式 (图 1A, 黄色阴影区域的算法)。对 DFS70 模式的误解如均匀、斑点或混合图案, 或反之亦然, 可能会对病人护理产生严重影响. 当前常用的协议如下: 临床实验室对疾病相关的阿纳斯和/或 DFS70/LEDGF 免疫方法进行了大量的固相验证性试验, 怀疑有密集的细斑点 (AC-2) 模式 (图1A)4,5。
该协议的目的是证明一个新的工程 HEp-2 IIF 基板 (HEp-2 ELITE/DFS70 击倒 (KO), 将被称为 HEp-2 精英) 的效用, 保留了传统 HEp-2 的所有优势, 以筛选阿纳斯同时区分 DFS70 模式与高置信度的 monospecific 和混合 ANA 阳性情况 (图 1B, 算法的黄色阴影区域)5。HEp-2 精华基板由未修饰的常规 HEp-2 细胞和无 LEDGF/p75/DFS70 抗原的工程细胞的近似混合物组成, 1:9 比为5。HEp-2 精英的 IIF 程序与传统的 HEp-2 方法相同。HEp-2 精华基板的独特配置不仅保留了常规 HEp2 的所有优点, 而且可以在样品的初始筛选或测试中区分均匀、斑点和混合反应模式 (图1B)5。患者血清在幻灯片上的反应 HEp-2 IIF 基质应稀释1:40 与筛选稀释或根据个别实验室标准。一个特定的反应在1:40 或更高的效价被认为是积极的。
在这项研究中, 阳性血清的均匀, 斑点, 丝, 核仁, 细胞质网状/医学和 DFS70 模式产生了中等阳性信号 (相对于负控制和阳性的 ANA 控制提供的套件) 在筛选稀释1:40 被选择用于模拟 HEp-2 基底 (常规和 HEp-2 精华) 的单特异性和混合型。1:40 稀释 DFS70 阳性血清与1:40 稀释的个别疾病相关的 ANA 模式控制 (均匀, 斑点, 丝, 核仁, 或细胞质网状-医学) 的各种比率 (control:DFS70 在 100:0, 80:20, 50:50,20:80, 和 0:100) 和评估的常规和 HEp-2 精英基板遵循标准 IIF 程序概述如下。

<table><tbody><tr><td>HEp-2 ELITE / DFS70-KO 12 Well Slide sealed individually in pouch</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240</td><td></td></tr><tr><td>ANA positive control</td><td>Trinity Biotech&amp;nbsp;</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>DFS70 antibody positive control</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240</td><td></td></tr><tr><td>Negative control</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Anti-human IgG FITC conjugate containing Evan&#039;s blue counterstain</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Buffer diluent for serum</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Mounting medium</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Coverslips&amp;nbsp;</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>ImmuGlo ANA HEp-2 Cells 12 well slide sealed individually in pouch</td><td>Trinity Biotech</td><td>part of kit# 1103, 1103-240</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials used in the study but not provided by the kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diagnostic fluorescent microscope</td><td>Nikon Eclipse 50i</td><td></td><td>Equipped with a spot dignostic camera, model 2.2.1</td></tr><tr><td>Incubation chamber</td><td>Trinity Biotech</td><td></td><td>provided for customers (no catalog #)</td></tr><tr><td>Coplin jar</td><td>n/a</td><td></td><td>General labware</td></tr><tr><td>Paper towels</td><td>n/a</td><td></td><td>General lab supplies</td></tr><tr><td>distilled/deionized water</td><td>n/a</td><td></td><td>General lab supplies</td></tr></tbody></table>

simultaneous distinction of monospecific and mixed dfs70 patterns during ana screening with a novel hep-2 elite/dfs70 knockout substrate

系统性自身免疫性结缔组织病的特点是循环核抗体 (ANA)。虽然有几种技术可用于 ANA 筛选, 间接免疫荧光 (IIF) 使用人上皮 cells-2 (HEp-2) 基质仍然是主要的和推荐的方法, 因为其优越的灵敏度。HEp-2 基板可以检测到多种模式产生的自身抗体绑定到各种蛋白质和核酸抗原分布在整个细胞核和细胞质的细胞。由于 HEp-2 基底上的正反应, monospecific 和混合模式的多样性也使报告的解释和准确性复杂化。最近受到极大关注的一个具体例子是, 由于自身抗体特异性地结合到一种叫做晶状体上皮细胞衍生生长因子 (LEDGF) 的蛋白而产生的致密精细斑点 70 (DFS70) 模式。缺乏明确的联系与特定的系统性自身免疫疾病和高患病率的健康人口已作出准确的解释 DFS70 模式的重要。使用传统的 HEp-2 基质, 准确区分 DFS70 模式与疾病相关模式具有挑战性。此外, 频繁共生的 DFS70 模式, 以及与疾病相关的模式, 如均匀, 斑点, 混合均匀斑点模式复杂的 IIF 解释。本文的目的是演示一种新颖的 HEp-2 IIF 基板的实用性, 它保留了传统 HEp-2 基板的所有优点, 同时提供了在两种情况下都能很好地区分 DFS70 模式的能力。monospecific 和混合的安娜阳性例子。新的基质更能揭开先前被 DFS70 模式掩盖的疾病相关的 ANA 模式。

HEp-2 ELITE/DFS70 高衬底保留了经典的 ANA 模式, 并有助于明确区分 DFS70 模式:
传统和工程细胞在 HEp-2 ELITE/DFS70 KO 衬底是相同的, 保存所有抗原除了 LEDGF/p75。该套件提供的负控制建立基线荧光信号。对 HEp-2 精英的均匀、斑点、丝、核仁和线粒体模式的积极控制产生与常规 HEp-2 IIF 基底上的预期模式相同的模式 (图 2A)。这些参考模式已详细描述了与每个模式的抗原和疾病协会以前4,29。表 1中提供了图 2A中观察到的模式的简要描述。
HEp-2 精英的 DFS70 模式:
在每个井中大约有10% 的细胞表现出一种致密的斑点图案 (被分配的命名: AC-2), 可以在相间的核上观察到, 染色质相关染色见于有丝分裂细胞核 (图 2B)。在传统的 HEp-2 基板上, 所有的细胞都将有这种模式时, 与 DFS70 阳性样品的反应。其余的90% 的 HEp-2 细胞有psip1基因编码的 LEDGF 抗原打掉, 因此不会产生一个信号或模式时, 与 monospecific DFS70 阳性血清 (图 2B) 的反应。如果10% 的 HEp-2 细胞有更明亮的荧光对应的 DFS70 模式和其余的细胞呈现额外的模式 (例如,精细斑点或核仁), 那么这表明存在的混合模式。对这种模式的更仔细的检查是必要的, 以确认所有的自身抗体的特殊性, 除了 DFS70 模式存在。为了证明 HEp-2 精华基板的能力, 一个血清样本小组创建了不同浓度的 DFS70 和 ANA 阳性控制血清和测试的传统和 HEp-2 精英基板使用标准的 IIF 协议 (图3,图 4,图 5,图 6,图 7)。
DFS70 Monospecific 和混合反应对传统和 HEp-2 精华基板的解释:
对于每个特定疾病相关的 ANA 模式 (图 3、图 4、图5、图 6、图7)、左大多数列上的 monospecific ana 模式和右大多数列上的 monospecific DFS70 模式可以观察到。中间的三列代表了疾病相关和 DFS70 模式阳性对照的各种比率。由此产生的混合模式在传统的 HEp-2 基板上很难辨别 (图 3、图 4、图 5、图 6、图 7、顶行)。然而, 以新颖的 Hep-2 精华基体, 在多数样品, 不同的强度之间的区别在未修饰和被设计的细胞之间是独特的, 不仅证实存在 DFS70 抗体 (当明亮的比率对较不亮的细胞是1:9) 但也揭示了第二个安娜模式。在 homogeneous-DFS70 或 speckled-DFS70 混合反应的情况下, 不可能满怀信心地预测正确的模式, 从而导致实验室选择运行一系列附加的检测 (图 1A)。在 centromere-DFS70 和 nucleolar-DFS70 混合反应的情况下, 怀疑 DFS70 阳性是非常具有挑战性的 (图 5,图 6)。与核仁和细胞质的网状/医学反应, DFS70 模式是不占优势的, 直到血清比例达到50%。在所有的例子中, HEp-2 精华基板可以呈现 DFS70 模式和底层的次级模式在更广泛的强度水平, 从而有助于更准确的 ANA 解释。
为了密切观察混合模式, 结果为各种50:50 组合的 ANA 和 DFS70 阳性控制被放大 (图 8) 的传统和 HEp-2 精英基板。它被鼓励观察两个界面和有丝分裂染色的所有 ANA 模式, 包括 DFS70 在 HEp-2 底物, 以提高解释和准确性的报告模式。然而, 对于混合模式, 划分细胞核所呈现的差异模式可能不足以促进准确的解释。红色箭头 (图 8) 表示常规和工程细胞中的有丝分裂细胞核。可以观察到, 在分裂细胞的混合 DFS70 反应不符合一套解释疾病相关的 ANA 模式的规则。黄色和蓝色箭头表示未修改和工程 (DFS70 KO) HEp-2 细胞核, 分别。在所有的组合 (图 8) 中, 观察到了未修改的细胞核染色 (约 10%) 和工程细胞核 (~ 90%) 模式在同一显微视场中的明显强度差, 这使得同时筛选和DFS70 模式的确认。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56722/56722fig1.jpg"> 图 1: 使用常规 HEp-2 和 HEp-2 ELITE/DFS70 方法的 ANA 筛选算法.(A) 示意图揭开了常规 HEp-2 IIF 在筛选阶段 (黄色阴影区域) 中识别 monospecific 和混合 DFS70 阳性模式的不足, 并无法排除二次疾病相关的 ANA 模式, 这可能被 DFS70 的图案所掩盖。此外, 目前产生的 DFS70 特定的固相验证性分析无法确认 monospecific DFS70 阳性, 从而导致对阿纳斯的额外验证性测试。(B) 示意图揭示了 HEp-2 精英的能力, 以筛选阿纳斯, 同时区分 monospecific 和混合 DFS70 反应, 并消除了需要固相 DFS70 确认化验。算法使用 HEp-2 精英显着简化了 ANA 筛选和减少所需的确认化验数量。<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56722/56722fig2.jpg"> 图 2: HEp-2 ELITE/DFS70 KO 衬底上的经典 ANA 和 DFS70 图案.(A) HEp-2 ELITE/DFS70 高衬底保留经典的安娜模式。经典的均匀反应和 DFS70 阳性反应产生使用的积极控制提供的套件。其他反应产生使用以前建立的阳性对照血清的斑点, 丝, 核仁和细胞质网状/医学协会反应5。(B) HEp-2 ELITE/DFS70 高衬底提供了常规和工程化的细胞, 便于 DFS70 模式的区分。示意图显示了产生的 DFS70 monospecific 反应和染色模式的界面和分裂细胞核的传统和工程 (DFS70-KO) 细胞。工程化的 HEp-2 细胞缺乏 LEDGF/p75/DFS70 抗原, 这使得对常规 HEp-2 基底上的 DFS70 自身抗体反应所掩盖的二次模式的同时检测。箭头 (黄色) 表示有丝分裂细胞核的例子。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56722/56722fig3.jpg"> 图 3: DFS70 和均匀的血清组合.DFS70 monospecific 血清被结合在不同比率 (100:0, 80:20, 50:50, 20:80 和 0:100), 并在常规和工程 (DFS70-KO) HEp-2 基板上测试的正均相模式控制。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56722/56722fig4.jpg"> 图 4: DFS70 和有斑点的血清组合.DFS70 monospecific 血清与积极斑点模式控制结合在不同的比率 (100:0, 80:20, 50:50, 20:80, 和 0:100) 和测试的传统和工程 (DFS70-KO) HEp-2 基板。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56722/56722fig5.jpg"> 图 5: 组合 DFS70 和丝血清.DFS 70 monospecific 血清结合了一个积极的丝模式控制在各种比率 (100:0, 80:20, 50:50, 20:80 和 0:100), 并在常规和工程 (DFS70-KO) HEp-2 基板上测试。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56722/56722fig6.jpg"> 图 6: 组合 DFS70 和核仁血清.DFS 70 monospecific 血清结合了一个积极的核仁模式控制在各种比率 (100:0, 80:20, 50:50, 20:80 和 0:100), 并在常规和工程 (DFS70-KO) HEp-2 基板上测试。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56722/56722fig7.jpg"> 图 7: DFS70 和细胞质网状/血清联合.DFS70 monospecific 血清与一个积极的线粒体模式控制结合在不同的比率 (100:0, 80:20, 50:50, 20:80, 和 0:100) 和测试的常规和工程 (DFS70-KO) HEp-2 基板。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56722/56722fig8.jpg"> 图 8: 混合模式 (50:50 比) 生产的传统和 HEp-2 的精英基板.两个基底之间的差异显露出来。红色箭头表示两个基底上的常规和工程细胞中的有丝分裂细胞核。对于这些组合, 通过在常规 HEp-2 基底上划分细胞核而提出的差分模式并不足以准确解释混合模式。可以观察到, 混合的 DFS70 反应的分裂细胞可以扰乱建立一套规则的解释疾病相关的 ANA 模式。黄色和蓝色箭头表示未修改和工程 (DFS70 KO) HEp-2 细胞核, 分别。在工程基板上测试的所有组合的结果: 观察到不被修改的细胞 (~ 10%) 和工程化细胞 (~ 90%) 之间的界面核模式之间的明显强度差, 这使得同时DFS70 模式的检测和确认。刻度线代表20µm.<a href="//cloudflare.jove.com/files/ftp_upload/56722/56722fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>安娜模式</td>
			<td colspan="2">描述</td>
		</tr>
<tr>
<td>均匀</td>
			<td colspan="2">整个细胞核荧光均匀, 呈弥漫性染色模式。</td>
		</tr>
<tr>
<td>斑点</td>
			<td colspan="2">离散, 粗到细斑点的荧光在整个细胞核。 细斑点和粗斑点图案已被描述。</td>
		</tr>
<tr>
<td>核仁</td>
			<td colspan="2">核仁染色为多个固体或有斑点的身体在细胞核内。子类型以前已被描述。</td>
		</tr>
<tr>
<td>丝</td>
			<td colspan="2">有限数的大斑点。反应性抗原隔离与凝聚染色体在有丝分裂细胞。</td>
		</tr>
<tr>
<td>细胞质网状/</td>
			<td colspan="2">细胞质呈粒状, 线粒体呈阳性染色。</td>
		</tr>
<tr>
<td rowspan="2">DFS70</td>
			<td colspan="2">•Conventional HEp-2 细胞: 在细胞核上观察到致密的斑点图案, 在有丝分裂细胞核上可见染色质相关染色。</td>
		</tr>
<tr>
<td colspan="2">•Engineered DFS70 KO 细胞: Anti-DFS70 抗体产生阴性染色</td>
		</tr>
<tr>
<td colspan="3">HEp-2 精英/DFS70 高有传统和工程 (DFS70 ko) 细胞约1:9 的比例。常规细胞产生典型的 DFS70 模式和工程细胞产生一个负面的模式, 当反应与 DFS70 monospecific/孤立的积极反应。</td>
		</tr>
</tbody></table>表 1: ANA 模式的描述。

Watch this Scientific Journal Video about Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate at JoVE.com

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

DFS70 自身抗体模拟常见的疾病相关的核抗体模式, 使准确的解释在使用传统的 HEp-2 基板的挑战。该协议描述了新的工程 HEp-2 基底优于传统的 HEp-2 在 ana 筛选和区分 DFS70 模式与高信心 monospecific 和混合 ana 阳性案件。

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

simultaneous-distinction-monospecific-mixed-dfs70-patterns-during-ana

Research

JoVE Journal

Immunology and Infection

35.5K 次观看.  A Trinity Biotech Company. DFS70 自身抗体模拟常见的疾病相关的核抗体模式, 使准确的解释在使用传统的 HEp-2 基板的挑战。该协议描述了新的工程 HEp-2 基底优于传统的 HEp-2 在 ana 筛选和区分 DFS70 模式与高信心 monospecific 和混合 ana 阳性案件。

视频: Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

双工数字PCR方法的同时定量<em&gt;肠球菌。</em&gt;和人的粪便相关HF183标记的水域

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

科研

环境科学

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

遗传学

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

发育生物学

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

免疫与感染

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

数字液滴PCR检测转基因按蚊种群中的Indels突变

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

对于资源有限设置一个负担得起的HIV-1耐药性监测方法

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

医学

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

黑腹果蝇基因座特异性核糖体 RNA 转录的单核苷酸多态性敏感 FISH 检测

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

生物学

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

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