Name: human serum anti-aquaporin-4 immunoglobulin g detection by cell-based assay
Uploaded: 2019-04-05T12:30:00
Description: Watch this Scientific Journal Video about Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay at JoVE.com

作者感谢国家科学基金会 (31600820)、吉林省卫生计划生育委员会 (原为 2016年 Q036号) 和吉林省科技规划项目 (编号) 的资助。20180520110JH)。

这一程序得到了吉林大学第一医院伦理委员会的批准, 并在大约 1, 500个科目上进行。
1. 患者注册和血液样本采集
<ol><li>将血清抗 AQP4 IgG 的实验室检测应用于临床患者的主要投诉和症状, 如下所示。也要进行身体检查。 视神经炎: 患者视力缺陷, 如视力丧失和视力下降。 急性骨炎: 患者可能出现瘫痪、感觉缺陷和自主功能障碍。 区域后发组综合征: 患者可能有不明原...

<ol>
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我们描述了一种广泛使用的检测人血清中抗 AQP4 IgG 的方法。抗 AQP4 IgG 与 NMOSD 密切相关, 建立可靠的抗 AQP4 IgG 检测方法对 NMOSD 的临床诊断至关重要。首先, 抗 AQP4 IgG 是 NMOSD 特有的。多发性硬化症也是一种免疫介导的中枢神经系统疾病, 与 NMOSD12有许多相似之处。然而, 抗 AQP4 IgG 在 NMOSD13中只有阳性。其次, 抗 AQP4 IgG 在 NMOSD 患者中很常见。约三分之二的 NMOSD 患者...

血清 AQP4 IgG 是诊断视神经髓炎视神经标志物的视神经标志物 (NMOSD)1。细胞基检测 (CBA) 是一种应用广泛的抗 AQP4 IgG 检测方法, 具有较高的灵敏度和特异性。这里介绍了 CBA 的详细协议。
AQP4 是一种水通道蛋白, 有六个跨越膜的单位和两个围绕一个水孔2的螺旋域。抗 AQP4 IgG 通过与目标 AQP4 结合参与 NMOSD 的发病机制, 其主要位于星形胶质细胞3的端部。研究表明, 在约三分之二的 NMOSD 患者中, 抗 Aqp4 igg 呈阳性。在最新的 NMOSD 国际诊断标准中, 抗 AQP4 IgG 被认为是一个核心诊断生物标志物5。在这方面, 建立可靠的检测人血清抗 AQP4 IgG 的方案, 促进 NMOSD 的临床诊断至关重要。
目前, 有各种抗 aqp4 igg 检测方法, 如 cba、组织基检测、酶联免疫吸附法和流式细胞术6、7。CBA 采用欧盟90细胞捕获抗 AQP4 igg, 这些细胞与人体 AQP4 转染。通过荧光二级抗体检测捕获的抗 AQP4 IgG, 然后通过显微镜观察。越来越多的证据表明, cba 比其他抗 aqp4 igg 检测方法更敏感、更具体, 6、7.根据 meta 分析, CBA 的敏感性和特异性分别为76% 和 99%, 高于组织基和酶联免疫吸附法 6.此外, 还对抗 AQP4 IgG 的诊断检测进行了多中心比较.共有来自15个欧洲诊断中心的193个研究对象注册了7人。采用四种不同的检测血清抗 AQP4 IgG7的方法。结果表明, CBA 比其他方法更敏感和具体7。由于 AQP4 是以两个主要异形 (AQP4-M1 和 AQP4-MO音二) 表示的, 抗 AQP4 IgG 捕获单元是用 AQP4-M1 或 AQP4-MOT 转染的。然而, 哪种类型的捕获细胞更好仍有争议。一项调查支持了以 aqp4-m1 为基地的 cba8, 而另一些调查表明, 以 aqp4-"3·23" 运动为基地的 cba 更好地7、9、10.然而, 由于 IGG 约束不具体, 基于 AQP4-"3·23" 运动的 CBA 可能会产生错误的阳性结果.Jarius 等人11. 11个国家报告说, aqp4-m1 和 AQP4----"3·23" 运动 cba 的抗性 Aqp4 igg 探测率没有明显差异。
总之, 血清抗 AQP4 IgG 是 NMOSD 的核心生物标志物。与其他抗 AQP4 IgG 检测方法相比, CBA 具有更高的特异性和敏感性。目前仍有争议的是, AQP4-M1-或基于 AQP4-23 的 CBA 更好。本文介绍了一种基于 AQP4-M1 的 CBA 的详细方案, 该方案可用于 NMOSD 的临床诊断和研究。

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	<tbody>
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			<td height="40" style="height:40px;width:185px;">Anti-aquaporin-4 IIFT</td>
			<td style="width:96px;">Euroimmun</td>
			<td style="width:105px;">FA 1128-2005-50</td>
			<td style="width:167px;">Contains biochip slides coated with AQP4-M1 transfected and untransfected EU 90 cells, fluorescein-labelled anti-human IgG, anti-AQP4 antibody as positive control, antibody negative sample, salt for PBS pH 7.2, Tween 20 and embedding medium.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CellSens Dimension</td>
			<td>OLYMPUS</td>
			<td>N/A</td>
			<td>photograph software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gel &#38; clot activator tube</td>
			<td>Improve medical</td>
			<td>623040202</td>
			<td>From a local Chinese company</td>
		</tr>
	</tbody>
</table>

human serum anti-aquaporin-4 immunoglobulin g detection by cell-based assay

抗水蒸汽蛋白-4 (AQP4) 免疫球蛋白 G (IgG) 是诊断视神经标志物的核心诊断视神经标志物的视神经功能性的视神经功能障碍。细胞基检测 (CBA) 是一种广泛使用的检测人血清抗 AQP4 IgG 的方法, 具有较高的灵敏度和特异性。简单地说, 血清抗 AQP4 IgG 是由 AQP4 转染细胞捕获的, 该细胞固定在生物芯片上, 然后由荧光标记的二级抗体检测到。荧光显微镜用于可视化荧光, 荧光强度由至少两名有经验的临床医生进行评估。可以结合抗 AQP4 IgG 检测结果、临床表现和神经放射学结果, 对 NMOSD 进行最终诊断。根据以往的研究, CBA 比其他抗 AQP4 IgG 检测方法更敏感、更具体, 可用于 NMOSD 的临床诊断和研究。该方法有局限性;例如, 目前还缺乏评估血清抗 AQP4 IgG 滴度的国际量表。本文介绍了一种利用 CBA 检测人血清抗 AQP4 IgG 的详细方案。

使用这里描述的程序, 血清中的特异性抗 AQP4 IgG 是可检测的。在手术过程中, 在反应场中添加了预稀释样品、阳性对照和负对照, 其中包含转染和未转染的区域 (图 2)。在转染区的负对照荧光主要表明二级抗体与生物芯片上转染细胞的非特异性结合 (图 3)。同质弱荧光可见 (图 3)。在阳性对照中, 在反应场中?...

Watch this Scientific Journal Video about Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay at JoVE.com

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

人血清抗水药物蛋白-4 免疫球蛋白 g 的细胞检测

细胞基检测是检测血清抗水品-4 免疫球蛋白 G 的一种广泛使用的方法。该方法可用于神经髓炎光谱疾病的临床诊断和科学研究。

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

根据最近国际神经骨髓炎视谱障碍诊断标准，抗AQP4 IgG被认为是一种核心诊断生物标志物。通过基于细胞的检测进行抗AQP4IgG检测，可促进临床诊断。因此，基于细胞的检测方法比其它检测方法更灵敏、更具体。并可应用于临床诊断和科学研究。首先，将患者的血清样本、生物芯片幻灯片、PBS 粉末和 TWEEN 20 从冷冻细胞检测试剂盒带到室温。然后，为每张生物芯片幻灯片准备超过 200 毫升的 PBS 洗涤缓冲液，其中含有 0.2% TWEEN 20。使用洗涤缓冲液对每种血清样品进行10倍的连续稀释。根据制造商的说明准备正负控制。将样品的10倍连续稀释、正控和负对，加入30微升，加入试剂盒上的5个单独反应场。握住生物芯片幻灯片的幻灯片，在不接触其反应场的情况下拆下其保护盖。将幻灯片放在试剂盒顶部，并在室温下孵育设置 30 分钟。用洗涤缓冲液轻轻冲洗生物芯片幻灯片。然后浸入装满 100 毫升洗涤缓冲液的 500 毫升烧杯中，并在室温下浸泡 10 分钟。从洗涤缓冲液中拿出生物芯片幻灯片。使用纸巾小心地从反应场外部擦去剩余的缓冲液。让滑梯风干一到两分钟。在暗室中，在试剂盒的每个反应场中加入25微升荧光二次抗体。将幻灯片放在试剂盒顶部，并在室温下孵育设置 30 分钟。用洗涤缓冲液轻轻冲洗生物芯片幻灯片。然后浸入一个500毫升的烧杯中，装满100毫升的新鲜洗涤缓冲液，并在室温下浸泡10分钟。从洗涤缓冲液中拿出生物芯片幻灯片。使用纸巾小心地从反应场外部擦去剩余的缓冲液。让滑梯风干一到两分钟。小心地将一滴嵌入介质添加到生物芯片幻灯片的每个反应场中。最后，用盖玻璃密封生物芯片幻灯片，同时避免气泡，然后继续成像。在感染区域观察到的阴性对照的微弱荧光强度表示二级抗体的不特异性结合。在受感染和未感染区域检测到阳性对照的荧光强度之间的比较表明，抗AQP4 IgG与感染细胞中AQP4M1的具体结合。抗AQP4 IgG阴性血清显示与受感染和未感染区域阴性控制类似的结合模式。阳性血清显示与受感染和未感染区域的阳性控制类似的结合模式。当荧光在感染区域异质增加时，样品被认为是抗AQP4IgG阳性的。荧光强度与血清中抗AQP4IgG滴度相关。当转染区域中只有少数细胞显示可检测到的荧光强度时，样本可能被认为是阳性的。当样本在感染区域的强度仅与未感染区域的强度均匀强时，应将其视为抗 AQP4 IgG 阴性。避免气泡是至关重要和困难的。要消除气泡，请首先使用移液器以避免液滴中的气泡。其次，在将生物芯片幻灯片涂到试剂盒上时，首先让幻灯片的一侧触摸液滴，然后缓慢地将幻灯片水平。此外，不要在整个实验中接触反应场。

Research

JoVE Journal

Immunology and Infection

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

确定使用血凝检测血清中的反应和滴度

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

科研

免疫与感染

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...