Name: anti-nuclear antibody screening using hep-2 cells
Uploaded: 2014-06-23T18:15:00
Duration: 13 min 1 s
Description: Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

我们感谢卡桑德拉科比用于执行IIF实验和卡罗尔布氏她的专家技术审查。

1，样本选择和准备基板<ol><li>从包装中取出试剂，让每一个项目，以达到室温。制备的试剂和根据方向插入稀释病人血清。注：NOVA精简版幻灯片有条形码，并可以很容易地集成到自动化系统与实验室信息系统LIS结合。此过程说明了手动滑动处理;高通量实验室，然而，可以选择自动滑动处理设备。 </li></ol> 2，添加控件和样品<ol><li>分配1滴阳性对照和1滴的阴性对照，以适当的滑动孔中。吸取20-25微升稀释的患者血清其余井。处理一个幻灯片的时间。 </li><li>将滑动（收费）用湿纸巾在底部染色容器。覆盖容器，并培育在滑动（S）处理30分钟。注意：在潮湿条件下将防止基板干燥。孔中的干燥会导致伪迹染色。在这段温育期，在患者的血清中的抗核抗体将结合到被固定在每个孔中的细胞的抗原。 </li><li>培养结束后，用洗涤缓冲液缓流冲洗掉的血清。为了避免损坏基板，并防止样品井之间交叉，角度滑动略，以避免直接引导流上的孔中。点击关闭多余的洗涤缓冲液，然后将滑入含有洗涤缓冲液约5分钟染色壶。 </li></ol>荧光共轭3。加<ol><li>一个接一个，从洗涤缓冲液去除幻灯片和轻轻拍打，以去掉多余的洗涤缓冲液。申请1滴荧光结合物（FITC标记的抗人IgG）的到各孔中。孵育的幻灯片在加湿容器30分钟，并且一定要更换染色容器盖。注意：对于ANA检测，使用与IgG Fc特异性结合的建议。所述缀合物是光敏感的，并且盖子将保护载玻片暴露于光中除了保持湿润的环境。在这段温育期，该偶联物将结合到病人的已结合到细胞抗原抗核抗体。这个偶合物结合导致荧光的孔中的存在。 </li><li>冲洗，洗如上所述的幻灯片。 </li><li>放置盖玻片在纸巾上，并应用安装介质中的连续线到盖玻片的底部边缘。 </li><li>从洗涤缓冲液去除每张幻灯片，然后点击幻灯片轻轻去除多余的洗涤缓冲液。触摸滑动的下边缘到盖玻片的边缘。 </li><li>轻轻降低滑动到盖玻片在盖玻片上的安装介质流向没有气泡形成在滑动的顶部边缘的方式。注：合作verslipping，包括使用安装介质的最佳量，是一种技术，需要实践来完善。 </li></ol>正面和负面的结果4。鉴定手动解读<ol><li>查看幻灯片用荧光显微镜，坐落在一个黑暗的房间。用20X或25X的目标进行扫描整个井，以评估细胞分布和荧光的均匀性</li><li>切换到40X的目标做出关​​于积极和模式的最终解释权。 </li><li>看看阳性和阴性对照。使用反应分级标准从1 +到4 +级阳性。注：阴性对照可能不会出现完全黑暗的，但往往会显示低水平的非特异性荧光。阳性对照将显示明亮的苹果绿色荧光在细胞核中（ 图1）。 </li></ol>自动化的解读注意：在additi上人工判读，负载和扫描幻灯片由NOVA查看自动荧光显微镜或其他自动数字成像仪;无需暗室是必要的。通过选择合适的滑动式创建项目后，仪器采集和存储单元的高分辨率数字图像在每个孔中。此外，NOVA查看测量荧光强度，并进行分类的结果为正或负，并且提供了模式识别为阳性样品。图像是由一个高分辨率的电脑显示器上的操作者观看，允许最终解释权归NOVA查看结果，修订和确认。报告可以在确定的业绩产生。数码显微镜应与有经验的实验室技术人员一起使用，因为它的目的是在结果的解释提供帮助。

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作者加布里埃拉洛科什，卡桑德拉科比，卡罗尔布氏，和安娜伊斯拉米是INOVA诊断的雇员。

Screening by HEp-2 cells is a critical first step in the diagnosis of patients with SARD. However, IIF methods lack harmonization. Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Despite these concerns, IIF remains the &#8220;gold standard&#8221; for ANA testing. HEp-2 cells contain a large variety of autoantigens, and provide the ideal substrate for the detection of these autoantibodies. In some laboratories, IIF has been replaced with solid phase screening methods such as multiplex assay or ELISA. The shortcoming of such tests is that they do not display the full range of antigens to be sufficiently sensitive. As a consequence, true positive patients may be missed which can have deleterious consequences In addition to the issues of treatment delay and wrong diagnosis, additional healthcare costs may occur due to the repetition of confirmatory tests or unnecessary diagnostic investigations. Given the inherent challenges to IIF, it is paramount to perform this technique properly to avoid subjectivity in results.
To ensure accurate interpretation and reporting of results for ANA screening, it is vital to use the highest quality substrate. When selecting a HEp-2 substrate, it is critical that the cells be optimized to express clinically relevant epitopes in their native protein state. Transfected or otherwise modified HEp-2 cell lines may not allow proper antibody-antigen binding. In addition, when several different autoantibodies are present simultaneously, recombinant cells may mask one or more patterns. High number of mitotic cells should also be present in the substrate. Adequate number of mitotic cells allows quick and accurate identification of patterns. 
In addition to the attributes of the substrate, the objective of this video protocol is to describe the IIF technique by showing critical steps such as the addition of sample, slide washing, addition of conjugate, cover slipping, and determination of positive and negative results. Results can be compromised if proper technique is not used for each of these steps. Proper washing is important to remove all unbound antibody. Some patients display very high amounts of autoantibodies and in these cases it is important to wash the serum from the wells such that it does not contaminate other wells. 
Although IIF has traditionally been a very labor intensive and subjective laboratory method, new technologies such as slide processors, barcoded slides and automated digital microscopy can greatly simplify the workflow, increase the consistency and reduce the sources of variability of interpretation.

术语抗核抗体（ANA）描述了多种与细胞的细胞核的成分包括DNA，蛋白质和核糖1,2反应的自身抗体。的Hep-2细胞，天然蛋白质阵列与数百抗原，提供了理想的底物为ANA 1的检测。全日空的人血清中检测是一个重要的筛选工具结缔组织疾病，IIF是全日空测试1的参考方法。最近，IIF在HEp-2细胞已被替换与抗原特异性免疫测定法和复用方法的一些实验室。由于担忧假阴性结果和缺乏透明度，临床医生，美国风湿病学院成立了一个特别工作组的结论是，IIF使用HEp-2细胞应该是“金标准”ANA筛查1。 由于主观ANA筛查的性质，HEp-2细胞的质量是在tegral到结果的准确和自信的报告。的大量有丝分裂的细胞的存在，最优细胞形态，足够汇合，以及相关抗原的表达是特别重要的。 IIF模式识别用作在患者以帮助诊断的重要工具。了解各种图案的意义使临床医生和实验室工作人员进行适当的后续测试，以确认诊断。例如，均相ANA模式可以发生在抗dsDNA或染色质的抗体的存在，并可能与全身性红斑狼疮（SLE）3相关联。从另一个方面，它最近已描述了所谓的致密的细斑点图案（DFS），其常见于常规样品的高达12％，主要是被用抗DFS70抗体有关。这些自身抗体，当隔离（无其他疾病特异性ANA）发现未与全身性自身免疫性风湿性疾病相关的&lt;燮&gt; 4-9。从而确证试验抗DFS70抗体可以帮助减少不必要的反射测试，提供了相当大的成本节约，并减轻病人的焦虑。 鉴于IIF是第一行筛选方法来检测自身抗体，它是最重要的，该用户能够理解如何试剂和组织基质的选择可影响结果。由于IIF技术本质上是主观的，重要的是，所用的试剂提供最高质量的结果。 该视频协议的这个目标是使用户熟悉执行IIF法，与ANA相关的常见模式所需的正确步骤，并引进新的进步，可以简化实验室工作流程和规范的结果。

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">NOVA Lite HEp-2 IgG (DAPI conjugate)</td>
			<td style="width:189px;">INOVA Diagnostics</td>
			<td style="width:135px;">708102</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NOVA View Instrument</td>
			<td>INOVA Diagnostics</td>
			<td>NV2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation</td>
			<td>INOVA Diagnostics</td>
			<td>LINK010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation License</td>
			<td>INOVA Diagnostics</td>
			<td>LINK001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barcode Scanner</td>
			<td>INOVA Diagnostics</td>
			<td>LINK019</td>
			<td></td>
		</tr>
	</tbody>
</table>

anti-nuclear antibody screening using hep-2 cells

对ANA检测风湿病立场声明的美国大学规定使用IIF作为全日空筛选1的金标准方法。虽然IIF是在专家手中一个很好的筛选试验，处理和阅读IIF的技术难点幻灯片 - 如劳动密集型幻灯片处理，人工读数，需要有经验的，训练有素的技术人员和使用黑暗的房间 - 使IIF法难以适应现代化，自动化实验室的工作流。 对高品质的ANA筛查的关键的第一步是仔细滑动处理。这个过程是劳动密集的，并且需要充分理解的过程中，以及关注细节和经验。 幻灯片阅读是通过荧光显微镜在黑暗的房间进行，由受过训练的技术人员谁是熟悉的各种图案，在细胞周期的背景下完成的和间期和分裂细胞的形态。但IIF是第一线筛检工具，可持续农业和农村，了解步骤正确执行此技术是至关重要的。 近年来，数字成像系统已经开发的IIF幻灯片自动读数。这些系统，如NOVA查看自动荧光显微镜，旨在简化日常IIF的工作流程。 NOVA查看获取并存储井的高分辨率数字图像，从而从解释分离图像采集;图像被视为一种解释型的高分辨率电脑显示器。它存储的图像以供将来参考，并通过提供对图像的荧光光强数据支持运营商的解释。它也预先进行分类的结果为正或负，并且提供了模式识别为阳性样品。总之，它省去了暗室，以及自动化和简化的IIF读写，程序纳克/口译工作流程。最重要的是，它增加了阅读器和读数之间的一致性。此外，与使用条形码的幻灯片，抄写错误是通过提供样品的可追溯性和积极的患者识别淘汰。这会导致增加病人的数据完整性和安全性。 此视频的总体目标是展示IIF程序，包括幻灯处理，识别常见的IIF模式，并引进了新的进步，以简化和协调这项技术。

当评估细胞染色结果喉癌Hep-2，五大核模式是最常见的：均匀，斑点，着丝粒，核仁和核点。这些模式的自身抗体结合至细胞核的特定成分的结果。虽然ANA检测是特异性的核结构，具有对细胞质结构自身抗体相关的细胞质图案也可以被观察到。在某些情况下，一些自身抗体可能存在于样品中，并且图像可能会显示为混合模式。 还有其它的，不经常观察到的，并且可以由IIF方法检测常细胞周期的特定模式;最近，所谓的密细斑点花纹具有特殊临床意义已经在某些ANA阳性血清描述。 均质模式要确定一个均匀的图案，扫描以及和识别有丝分裂或分割细胞。有丝分裂细胞的染色质凝集（ 图2，橙色箭头）显示出固体的均匀荧光这往往比在静息细胞nuclei.In均质模式，休眠细胞表现出均匀，整个核的弥漫性荧光（ 图更加明显2，白箭头）。这个特征模式往往是抗dsDNA抗体10的结果。 斑点型在粗斑点图案，有丝分裂的细胞显示没有染色简明的染色体区域（ 图3，橙色箭头）。在静息细胞中表现出粒状荧光在整个核。核中的斑点可以被定义为粗或细。粗斑点图案往往是抗Sm和抗RNP 11的结果。 在细斑点花纹，静息细胞显示细腻或弥漫性斑点整个细胞核，通常在统一ðistribution（ 图4中 ，白色箭头）。微细斑点图案往往是抗SSAand抗SSB 12的结果。 密集的细斑点型它现在认识到，致密细斑点图案（DFS）是常见的常规试验，并且多达12％的样品是阳性抗DFS70自身抗体。然而，这些自身抗体，当孤立地发现，不与全身性自身免疫性风湿性疾病4 -6关联。这些抗体多见于健康人，病人承载无关的可持续农业和农村6种疾病。验证性测试抗DFS70抗体可以帮助减少不必要的反射测试，提供可观的成本节约，并减轻病人的焦虑。 由于DFS模式是难以识别和ANA阳性会导致焦虑，以患者和医生，这是强烈建议鉴定​​后，进行确证试验<html> fying模式暗示抗DFS70抗体的澄清全日空的临床意义positivty 5,6。 在这个模式中，有丝分裂细胞（ 图5，橙色箭头）显示中期染色体阳性染色斑点，而静息细胞的细胞核（ 图5，白箭头）展览整个细胞核均匀分布的细小斑点。 着丝粒模式要识别着丝粒的模式，扫描井，并确定有丝分裂或分裂的细胞。在有丝分裂细胞（ 图6，橙色箭头），这些离散斑点成为什么经常被描述为一个“中期栏”密切相关。在着丝粒的模式，休眠细胞（ 图6，白箭头）显示整个细胞核分布约40-60离散斑点。着丝粒模式是抗着丝粒蛋白A中的结果，B，C抗体13。 步“&gt;核仁模式一些核仁图案被漫细胞质染色中的有丝分裂细胞中（ 图7，橙色箭头）和负极染色体区域相关联，而其他nucleolear图案显示染色体区域的阳性染色。核仁模式与核仁的同质或斑点染色（ 图7，白色箭头）相关联，随着弱斑点或核质的均匀染色。这些模式都与抗RNA聚合酶III相关联，抗核仁纤维，anti-Th/To和anti-PM/Scl抗体14,15。 核圆点图案核点图案与负中期有丝分裂的细胞（ 图8，橙色箭头），并与在静止细胞的细胞核几个离散斑点（ 图8中白色箭头）相关联。这个特征模式往往是SP-100，PML，或P80科林自身抗体的结果。这些抗体可以作为<html> sociated原发性胆汁性肝硬化和自身免疫性肝炎16。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> 图1：阴性对照（左）：电池显示低级别非特异性荧光，但没有具体的核染色阳性对照（右）：细胞显示苹果绿核荧光。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> 图2。同质格局。有丝分裂细胞（orangearrow）表明固体荧光。静息细胞显示均匀，弥漫性染色（whitearrow）。 “宽度=”500“/&gt; 图3。粗斑点型。有丝分裂细胞（橙色箭头）显示阴性染色。静息细胞表现出明显的斑点染色（白色箭头）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> 图4。细斑点图案。有丝分裂细胞（橙色箭头）显示阴性染色。静息细胞表现出明显的细斑点染色（白色箭头）。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> 图5。密细斑点花纹。有丝分裂细胞（橙色箭头）显示出细粒状固体染色。静息细胞表现出非常精细的，弥漫性斑点状染色（whitearrow）。 图6。着丝粒的模式。有丝分裂细胞（orangearrow）显示均匀，离散斑点。静息细胞（whitearrow）显示每个细胞核40-60离散斑点。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" /> 图7。核仁格局。有丝分裂细胞（orangearrow）出现大簇颗粒染色。静息细胞nucleishows在核仁荧光（whitearrow）。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" /> 图8。核圆点图案。有丝分裂细胞（orangearrow）出现负值。静息细胞显示了几个DISCR在细胞核中（whitearrow）ETE斑点。此外，nucelar点图案可以与自身抗体线粒体抗原（红色箭头）相关的细胞质染色共存。

Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Anti-Nuclear Antibody Screening Using HEp-2 Cells

间接免疫荧光（IIF）的测定法在传统上被用于抗核抗体（ANA）的人血清中的检测。这些抗体的存在可以在全身性自身免疫性风湿病（SARD）帮助诊断。该协议演示了如何有效地进行IIF技术，准确检测这些自身抗体。

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 ...

anti-nuclear-antibody-screening-using-hep-2-cells

Research

JoVE Journal

Biology

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...