Name: a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes
Uploaded: 2014-03-25T13:45:00
Description: Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

这项工作是由美国国立卫生研究院资助R01-GM-098977和R21-AI-098122到CBK和KEG支持。 RSS是由塞耶创新项目奖学金从工程学院塞耶支持的，部分由卢斯基金会奖学金和部分。

四个主要业务包括肽-MHC II结合试验：1装帧：测试肽标记的对照肽争夺可溶性MHC II类蛋白的溶液相结合。结合测量在很宽的范围内测试肽浓度。 2 - 捕捉：在结合反应接近平衡，肽-MHC II类分子复合物被捕获并与未结合的肽和蛋白的构象依赖性的认可与固定化抗体分离。 3，检测：捕获控制肽是利用时间分辨荧光定量检测。 4分析：光谱数据进行处理，绘制和分析，以确定测试肽与MHC II类蛋白的剂量...

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生物疗法已经确立了自己作为现代医学的基石，相当于2012年32四个五大畅销的药物。在生物制药行业呈现出持续增长几年6，以及新药的不断开发，以及生物仿制药的出现，扩大了生物制药管道。展望未来，评估和减少蛋白质治疗的免疫原性将成为早期生物治疗性发展的组成部分。为了促进这一进程，生物技术可以利用自己的计算方法为表位预测16-22以及旨在减少免疫原性?...

蛋白质是增长最快的类治疗药物6，生物治疗和管道的快速扩张已经越来越注意与开发蛋白质药物和使用相关的挑战。一个独特的代价源于以下事实：在一个健康和功能的免疫系统中，所有的细胞外蛋白质是由抗原呈递细胞（APC）进行采样。一旦被APC内化的蛋白质裂解成小的肽片段，以及推定的免疫原性片段被加载到II类主要组织相容性复合蛋白质（MHC II）的槽。肽-MHC II类复合物，然后显示在APC的表面上，并且真正的免疫原性肽，称为T细胞表位，形成三元MHC II类分子-肽-T细胞受体复合物与同源CD4 + T细胞表面受体7。这个关键的分子识别事件启动一个复杂的信号级联放大，这导致T细胞活化，细胞因子的释放S，CD4 + T细胞介导的​​B细胞成熟，并最终生产流通结合并清除违规的外源蛋白的IgG抗体。因此，免疫原性蛋白可能通过识别组成T细胞表位和变异负责MHC II类分子复合物的形成关键残基进行脱敏。它承担但指出，该T细胞表位可以是很多，大致整个免疫原性蛋白分布，和广大的表位删除突变都可能导致蛋白质功能或稳定性的意外丢失。因此，工程脱敏生物疗法可能是一个复杂和技术上具有挑战性的目标，但是存在成功的T细胞表位缺失项目3,5,8-12的几个例子。不像嫁接型“人性化”，这在很大程度上仅限于抗体疗法，表位缺失可应用于基本上任何蛋白质靶无论序列，结构，功能，或homologo的可用性我们人类的支架。第一步，采取这种方法是识别嵌入靶蛋白序列中的关键肽表位。 使用合成肽和重组人MHCⅡ类分子高通量生化分析可以提供快速的初步见解表位识别和缓解1,3-5。这些类型的ELISA检测可以是一个强有力的补充其它蛋白质/疫苗设计和开发工具。例如，一个公认的实验方法，以表位作图依赖于时间，劳力和资源密集的体外细胞增殖测定15。简言之，将靶蛋白的一级序列首先被分成重叠肽的面板中，常常15 - 聚体与12个残基相邻的肽之间重叠。肽面板化学合成和每种肽的免疫原性是在几个雇用外围布卢不同免疫1测试从人供体13,14隔离数字单核细胞（PBMC）。为了提供更大的信心，结果，肽通常在重复与PBMC从50个或更多不同的捐赠者进行测试。在情况下，去免疫化是最终目标，工作是由需要产生突变肽的附加 ​​板和引入任何deimmunizing突变成全长蛋白为后续功能分析10日前 PBMC中检测测试新的肽面板进一步加剧。虽然这些细胞试验仍然是金标准评估在人类患者的免疫原性潜力，这样的穷举的方法的效率可能通过预过滤推定的免疫原性表位使用快速和高通量MHC II类肽结合试验加以改进。 同样，生化肽-MHC II类分子结合分析可以预测，在硅片的方法相结合，从根本上加速了表位识别的过程。存在各种用于T细胞表位预测的计算工具;例子包括ProPred 16，MHCPred 17，SVRMHC 18，ARB 19，SMM-20对齐，NetMHCIIpan 21以及专有工具，如EpiMatrix由EpiVax 22。同样地，表位预测器最近已结合其他生物信息学和分子建模工具，得到目的是减轻deimmunizing突变可能会破坏蛋白质的结构和功能23-26风险集成蛋白去免疫化的算法。虽然一些抗原表位的预测已被证明是相当准确27,28，计算结果总是需要实验验证。快速，高通量和成本有效的实验方法最适合作为在硅片表位预测的初步过滤。 与此类似，表位的预测可以带动抗原选择反向vaccinoloGY 29,30。例如，在生物信息学的进步已经产生了全基因组的屏幕，迅速地识别在从病原体蛋白质组提取全蛋白质或肽表位的形式的候选疫苗。虽然这种技术能够应用在重塑发现疫苗的保护和发展，它引入了新的挑战中的免疫原性候选疫苗难以控制大型列表的形式。高通量肽-MHC II类分子结合试验可以通过量化肽结合亲和力和结合在多个MHCⅡ类等位基因滥交引导表位的选择。由于与蛋白质脱敏，这样的实验方法最终需要验证的有前途的疫苗导致计算预测。 这里，缩放，以384孔格式的肽-MHC II类分子结合测定法进行说明。该协议是高度并行和相比于以前描述过的96孔板格式1,3-5降低了试剂的成本减少75％。使用单一的液体。ð搬运机器人，这种方法允许一名研究人员能够轻松地分析，一式三份大约90测试肽在一定范围内八个浓度和四个MHCⅡ类等位基因类型，在不到48小时。这篇文章描述了一个384孔ELISA板为7的实验肽对1 MHC II类等位基因的分析的设置，但扩散片计算器被提供作为辅助材料，以便容易地在实验扩展到任何数量的期望的肽和/或MHC的II类分子。

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			<td height="35" style="height:35px;width:587px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:357px;">Sigma</td>
			<td style="width:217px;">221732</td>
			<td></td>
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			<td height="35" style="height:35px;">Citric Acid</td>
			<td>Sigma</td>
			<td>C1901</td>
			<td></td>
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			<td height="35" style="height:35px;">Dibasic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trizma HCl</td>
			<td>OmniPur</td>
			<td>9310</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Tween-20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">100% DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Octyl-&#946;-D-Glucopyranaside</td>
			<td>Fischer</td>
			<td>29836-26-8</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Pefa Bloc SCF</td>
			<td>Roche</td>
			<td>1158591600</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dulbecco&#8217;s 10X Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14200-166</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Assay Buffer</td>
			<td>Perkin Elmer</td>
			<td>4002-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Enhancement Buffer</td>
			<td>Perkin Elmer</td>
			<td>4001-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Europium Labelled Streptavidin</td>
			<td>Perkin Elmer</td>
			<td>1244-360</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">L243 anti-HLA-DR antibody</td>
			<td>Biolegend</td>
			<td>307602</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Biotinylated tracer peptides</td>
			<td>21st Century Biochemicals</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Test peptides (1-4mg, 85% purity)</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Purified HLA-DRB1 monomers (non-biotinylated)</td>
			<td>Benaroya Research Institute*</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">384-well white EIA/RIA plate</td>
			<td>Thermo</td>
			<td>460372</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Polypropylene 384-well plate</td>
			<td>Costar</td>
			<td>3656</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Film, AxySeal, 80 &#181;m, ELISA Applicator&#160;</td>
			<td>Axygen Asicentific</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">MilliQ Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Epimotion Liquid Handler (or similar)</td>
			<td>Eppendorf</td>
			<td>5075</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Select TS Plate Washer (or similar)</td>
			<td>BioTek</td>
			<td>405</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SpectraMax Gemini Microplate Reader (or similar)</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4" height="98" style="height:99px;width:1161px;">*Recombinant human MHC II molecules can be obtained from the Benaroya Tetramer Core Laboratory. See: https://www.benaroyaresearch.org/our-research/core-resources/tetramer-core-laboratory</td>
		</tr>
	</tbody>
</table>

a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes

生物化学测定重组人MHC II类分子可提供快速，定量分析上市免疫原性表位的识别，缺失或设计1,2。这里，肽-MHC II类分子结合测定法进行缩放，以384孔格式。按比例缩小的协议降低了试剂的成本减少75％，并且比以前描述的96孔方案1,3-5更高的吞吐量。具体而言，实验设计允许多达15个肽对每384孔ELISA板1 MHC II类等位基因的健壮和可重复性分析。使用单一的液体处理机器人，这种方法允许一个研究者来分析，一式三份约90测试肽在一个范围内的8浓度与4 MHC II类等位基因的类型在小于48小时。其他蛋白质去免疫或疫苗的设计和开发领域的工作可能会发现协议是在促进自己的工作非常有用。特别是，一步一步的指示和视觉格式朱庇特应允许其他用户能够快速，方便地建立这种方法在自己的实验室。

阴沟肠杆菌 P99β-内酰胺酶（BLA）（基因ID＃X07274.1）的成熟肽序列与ProPred 16分析假定的肽基料，以MHCⅡ类等位基因DRB1 * 1501（ 表4）。 ProPred确定117九聚肽具有强制性P1锚定残基（ 即一个M，L，I，V，F，Y或在 ​​位置1，这是需要MHC II类分子结合31瓦）。以5％的门槛，只肽分值大于或等于2.6有可能粘合剂。因此，在5％的门槛，只有排名前11肽预测结合MHCⅡ?...

Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

生物化学测定重组人MHC II类分子可提供快速，定量分析上市免疫原性表位的识别，缺失或设计。这里，缩放到384孔板的肽-MHC II类分子结合测定法进行说明。这种成本效益的格式应该证明在蛋白质去免疫和疫苗的设计和开发领域非常有用。

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Research

JoVE Journal

Biology

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Saccharification Assay for Lignocellulosic Materials

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Quantitative Fitness Analysis Workflow

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

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

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High Throughput Microinjections of Sea Urchin Zygotes

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

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

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 ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...