我们感谢加拿大西部大学的肖恩. 李和邢力对 SPOT 阵列产品的帮助。我们感谢在组织相容性核心的工作人员和西北大学综合移植中心提供样品服务。这项工作得到了部分支持, 西北纪念医院的辅助委员会, 并由一个教师启动基金由西北大学提供给 jj。

此处描述的所有方法都已获得西北大学机构审查委员会 (IRB 协议号: STU00104680) 的批准。 图 1 说明了协议的总体工作流. 
 1. 生物分析捐赠人和接受者 hla 序列 <ol> <li> 从 IMGT/hla 数据库中检索序列 15 。
	<ol> <li> 获取器官捐献者和他/她的接收者的 HLA 打字报告. 
		注意: 确保使用适当的程序来保护保密病历。通常情况下, 机构审查委员会 (irb) 批准的研究协议或实验测试是根据机构内部评级准则。如果 HLA 报告来自 pcr 打字 (高或低分辨率) 或 sequencing-based 打字 (SBT), 直接进入步骤1.1.3。如果序列是从基因组/HLA 测序中获得的, 如果它们是完整的, 则直接使用这些序列。有时, 当仅有不完整的键入或排序结果可用时, 请执行附加步骤1.1.2。通过在步骤1.1.2 中描述的 web 工具来分配 #34; 丢失和 #34; 等位基因. </li>
		<li> 在 web 链接 http://www.ebi.ac.uk/ipd/imgt/hla/ambig.html 后打开不明确的等位基因组合, 并使用 and #8220 进行搜索; 模糊等位基因组合搜索工具和 #8221; 函数检索假设等位基因。然后, 继续步骤1.1.3。输入等位基因的名称. </li>
		<li> 打开 IMGT/HLA 等位基因查询表, 在下面的 web 链接-http://www.ebi.ac.uk/ipd/imgt/hla/allele.html, 并输入主题和 #8217; s 等位基因名称 (每个单独的, 如 图 2 ) 进入和 #8220; 搜索#8221; 盒子点击 #8220; 搜索等位基因现在和 #8221; 按钮。HLA 等位基因名称应使用标准命名系统 ( 即 、A *、A*01、A*01:01:01:01 和任何以前的指定, 如 A*01010101). </li>
		<li> 查找屏幕上显示的匹配等位基因名称。单击 "等位基因" 按钮. </li>
		<li> 复制 #34;P rotein 序列和 #34;, 并将其粘贴到文本文档中的标题行和 #34; #62; 等位基因名称和 #34;。有效地, 文本文档是以 FASTA (快速) 的等位基因的名称和序列格式. </li>
	</ol> </li> <li> 执行基因多个对齐的施主和受体/患者等位基因。
	<ol> <li> 一个基因 ( 即, DQB1*03:01:01:01) 一次复制和粘贴所有四位基因的捐助者和患者 FASTA 序列 (两个从捐赠者和两个病人) 到一个合并的文本文件。在这一点上, 它是重要的差异表示, 从患者等位基因名称的供体基因名称 (选项包括差异使用的上部与较低的情况; 或创建一个区别的前缀或后缀扩展到每个基因的名称分别标记捐赠者 vs。患者等位基因)。序列的输入顺序是不重要的, 因为跟随对齐顺序将根据任意对序列的相似性的水平被洗牌. </li>
		<li> 以 FASTA 格式复制和粘贴序列 (如图 3 ), 从上到 #8220; 输入您的输入序列和 #8221; 在群集欧米茄网站上的框: http://www.ebi.ac.uk/Tools/msa/clustalo/. </li>
		<li> 单击并 #34; 提交您的工作和 #34; 使用参数的标准设置: 例如, #34;P rotein 和 #34;; #34; Clustal w/数字和 #34;。通过单击和 #34 来执行对齐; 提交和 #34;。四序列的对齐方式显示在屏幕上 ( 图 4 ). </li>
	</ol> </li> <li> 标记特定于捐助者的不匹配. 
	注意: 应该特别注意的是, 这里给出的位序列是基于 HLA 类型的结果, 而不是测序。因此, 有可能错过特定的捐助者-受援对的相关序列变化的风险。随着越来越多的移植中心采用精确的高分辨率分型和直接 DNA 测序, 这个潜在的问题将会得到缓解。
	<ol> <li> 将对齐文本复制并粘贴到文本文档中并创建新文档。如果对齐的文件的格式被打乱, 请通过更改字体样式和大小来修复格式: 始终使用和 #34; 信使新的 #34; 小字体大小以适合行。在解决格式问题后保存文档. </li>
		<li> 手动检查对齐的序列, 以确定所有不匹配的残滓属于唯一的捐赠者。使用区分字体颜色标记这些特定于施主的残滓. </li>
		<li> 在捐赠者和 #39 的所有字母下划线, 扩展14残留的序列都 up-and 下游标记的供者特定的不匹配 (计算为15氨基酸肽减去1的不匹配的残余). </li>
	</ol> </li> <li> 在重叠的系列中派生 15-滨海肽序列. 
	注: 选择 15-氨基肽的原因是基于一般知识的典型表位是4-9 氨基酸的长度。因此, 当我们应用一个和 #34; 移动窗口和 #34; 步骤4氨基酸的步长 ( 图 1 ), 我们选择了一个15氨基酸长度留下了 15-4 = 11 氨基酸重叠在任何相邻肽之间的一系列。从理论上讲, 这11氨基酸重叠足以覆盖所有长达9氨基酸的免疫表位, 因此不会被无意和 #34; 分裂和 #34; 在一半, 只有部分序列在阵列上。
	<ol> <li> 使用带下划线的序列作为模板, 在序列中连续导出短15氨基酸序列, 该系列中的任意两个相邻序列之间的4残差重叠. </li>
		<li> 将这些15氨基酸序列复制并粘贴到电子表格 ( 图 5 ) 的列格式中, 同时加上包含相应的施主等位基因名称的符号列。附加的列, 包括 aa 残留位置可能是有用的. </li>
	</ol> </li> <li> 重复从1.1.3 到1.3 的步骤. 对每个 HLA 等位基因 ( 即, A, B, C, DQA1, DQB1, DRB1 和 DP) 的捐助者和收件人对得出 15 aa 短序列的捐助者. </li>
	<li> 将所有列复制并粘贴到主电子表格中, 以创建来自捐赠者所有等位基因的 15 aa 序列的长连续列。记下文档中的总行数 (用于多肽序列). </li>
</ol> 2。自定义阵列布局和生产设计 <ol> <li> 生成具有相应等位基因召唤的多肽序列的电子表格。该阵列有20行和30列, 可以容纳 20x30 = 600 点的多肽。根据在1.5 步中记录的肽的总数, 从一个特定的捐赠者, 并根据我们过去的经验与两个例子, 我们曾尝试在刘 et al 。 17 , 600 点阵列可以容纳由2独立的移植案例生成的所有序列。
	<ol> <li> 合理规划最大的 e常数的方式, 以适应组的多肽在600点格式的数组. </li>
		<li> 如果多个主题可以容纳在一个整数数组中, 则在第一个 "捐助者" 和 "#39" 之后插入空行, 以匹配可除以30的行总数 (数组中一行的斑点数). </li>
		<li> 紧接在第一组序列的最后一个空行之后, 从第二个移植案例中粘贴到序列内容的整个列中. </li>
		<li> 重复这些步骤, 直到填充了阵列, 并且在不超过600点的情况下也不能添加任何事例。如果将整个空行留在底部, 并 #34; 移动和 #34; 在两个施主集之间放置空行。这宽空间在案件之间留下在综合化的完成以后做切开膜容易-跟随步骤3.2。下面. </li>
	</ol> </li> <li> 在数组布局的上下文中对多肽序列进行编程。现场合成器的节目采取序列的文本格式 (一列一多肽序列), 可以直接地获得通过保存得到的电子表格从步2.1.4 作为一个简单的文本文件 (不额外专栏 (s) 为等位基因名字, aa位置 等 ). </li>
	<li> 通过现场合成器运行自动肽阵列合成。在 Kudithipudi et al. 21 中详细介绍了该合成的整个操作。请注意, n-芴甲氧羰基合成两个阵列需要4-5 天. </li>
</ol> 3。探针和再血清从个人移植接受者的时间序列. 
 注意: 600 点膜阵列具有尺寸 ~ 7 厘米 x 13 厘米。经过合成, 这些阵列可以在室温下作为干膜保存至少两年, 当屏蔽直接光。避免过度折叠的膜, 以保持其长寿, 重复使用. 
<ol> <li> 用乙醇水化膜阵列, 并可视化胭脂红 S 染色的多肽斑点. 
	<ol> <li> 水化在 li et al. 中优化的分步过程中携带多肽的膜阵列 24 . <ol> <li> 将阵列膜浸入100% 乙醇的 20 mL 中. </li>
			<li> 添加20毫升蒸馏水稀释溶液到50% 乙醇, 室温孵育15分钟. </li>
			<li> 将浸泡解决方案更改为40毫升100% 水三次, 每次孵育15分钟. </li>
			<li> 在适当的工作缓冲区中清洗, 例如, 20 毫升的 TBST (三缓冲盐水与 0.1%), 三次为5分钟. </li>
		</ol> </li> <li> 胭脂红的数组的着色以可视化合成多肽 <ol> <li> 将20毫升好胭脂红的溶液直接添加到水合阵列中, 并在三十年代以颤抖的方式孵育. </li>
			<li> 连续在膜上运行蒸馏水以 de-stain 背景胭脂红的颜色。在这个过程中, 红颜色的多肽斑点可能变得可见. </li>
			<li> 将数组的各个部分分别为每个捐赠者, 小心地在阵列的各个部分之间进行切割。标记每个数组的方向. </li>
		</ol> </li> </ol> </li> <li> 前块阵列. 
	<ol> <li> 将20毫升的5% 脱脂牛奶中的膜块溶解在 TBST 缓冲液中。这个牛奶的解决方案将进一步 de-stain 胭脂红的颜色。多次更换牛奶缓冲液, 以达到最清晰的多肽图像. </li>
		<li> 为了保持记录的目的, 请使用 hand-held 照相机 (例如, 在 图 6 ) 中拍摄阵列的胭脂红 S 映像. </li>
		<li> 将5% 脱脂牛奶中的膜继续阻塞在4和 #176; C 隔夜或室温下为2小时, 摇晃. </li>
	</ol> </li> <li> 使用移植抗血清来孵育数组. 
	注意: 应注意的是, 一种称为前或钩效应的现象可能是由于血清抗体分析中的补体干扰引起的。为了绕过这个问题, 可选择的血清预处理步骤可以考虑 EDTA 或热失血清样品。
	<ol> <li> 删除阻塞缓冲区, 并在第二天每天三次的情况下用20毫升的 TBST 清洗膜, 每次5分钟. </li>
		<li> 添加20和 #181; 接受者的粗血清为20毫升的 TBST 缓冲液中的2.5% 牛奶, 室温下用膜孵育2-3 小时。请注意, 在这首轮的探索, 建议在时间序列的最后移植血清 (或可能最敏感的血清) 首先使用。这是基于这样的假设: 早期的标本, 特别是从前的时间点, 有较少的品种的抗体, 往往随着时间的推移发展。这样, 任何可能的信号干扰的 #34; 结转和 #34; 在探测回合之间可以清楚地区别于真正发达的同种反应, 由于免疫反应对移植. </li>
	</ol> </li> <li> 使用20毫升的 TBST 清洗阵列, 并用二次抗体孵育. 
	<ol> <li> 每次清洗膜三次10分钟. </li>
		<li> 孵育与山羊人 IgG-HRP (辣根过氧化物酶) 二次抗体在1:10,000 稀释在 TBST 缓冲补充与1% 牛奶为另外 2 h. </li>
	</ol> </li> <li> 清洗并开发印迹。
	<ol> <li> 每次用 TBST 清洗膜三次10分钟. </li>
		<li> 使用5毫升的鲁米诺溶液进行增强化学发光 (ECL), 用5毫升的过氧化物溶液新混合, 开发膜 (1 分钟). </li>
		<li> 使用合适的成像器 ( 即 ChemiDoc 成像系统或 Azure C600) 可视化 ECL 信号. </li>
	</ol> </li> <li> 扫描并量化印迹. 
	<ol> <li> 保存已开发的图像 ( 图 7 、下图) 并对光斑强度进行量化. </li>
	</ol> </li> </ol> 4。在临床时间序列上比较抗血清反应性. 
<ol> <li> 剥离数组. 
	<ol> <li> 在这一点上, 始终保持膜湿. </li>
		<li> 用20毫升的商业剥离缓冲器在37和 #176 上剥离膜; C 为20分钟, 然后用 TBST 三次清洗膜, 每次10分钟。然后重复阻塞 (步骤 4.2) 和探测 (步骤 4.3) 步骤, 使用不同时间点的患者血清. </li>
	</ol> </li> <li> 块剥离膜. 
	注意: 在剥离后, 膜可以被重复使用, 以便在一个时间序列中对同一患者的不同血清进行另一轮探测。
	<ol> <li> 使用5% 的牛奶缓冲来阻止膜 (请参见步骤 3.2). </li>
	</ol> </li> <li> 从同一时间序列中再不同的抗血清 (重复步骤从 3.3-3.6; 示例在 图 7 中, 上图). 
	注意: 剥去的阵列可以用来再另一个血清标本。由于多肽与膜的支撑基体共价共轭, 我们表明该阵列可重复使用多达20轮剥离和 reprobing 循环, 而不会失去性能rmance. </li>
	<li> 数组的长期存储. 
	<ol> <li> 将存储在 TBS 缓冲器中的膜, 以 0.02% w/w 叠氮化钠作为防腐剂。使用密封塑料袋进行长期贮存。在4和 #176; C 在保护之下免受直接光, 湿膜可以被存放至少2年. </li>
	</ol> </li> </ol> 5。数据获取和分析 <ol> <li> 手动注释正抗原肽。
	<ol> <li> 定位显示阳性抗体信号的斑点, 并确定它们的每个网格位置. </li>
		<li> 在主工作表中突出显示正肽的对应行. </li>
	</ol> </li> <li> 检索主工作表中列出的多肽序列 ( 图 7 , 底部列表)。根据步骤1.4.1 中概述的设计原则, 相邻的序列点具有明显的重叠顺序 (15-4 = 11), 因此反应表位可由多肽在连续序列中共享。
	<ol> <li> 突出显示连续发生的任何正点. </li>
	</ol> </li> <li> 确定系列中每个肽的最小表位长度。
	<ol> <li> 根据活性肽的重叠片段突出显示任何可能共享的表位序列. </li>
	</ol> </li> <li> 结构模型抗原表位。
	<ol> <li> 从以下 web 链接中找到的蛋白质数据库获取原型 HLA 晶体结构: http://www.rcsb.org/pdb/home/home.do. </li>
		<li> 在 Pymol 中显示原型 HLA 结构 (从 www.pymol.org 下载). </li>
		<li> 映射和 #34; 或模型通过突出显示反应性肽序列 ( 图 8 ) 发现 anti-donor 表位与原型 HLA 分子的3D 结构。单击 #34;D isplay 和 #34; 然后 #34; 背景和 #34;, 选择和 #34; 白色和 #34;。要保存图像, 请转到 #34; 文件和 #34; #34; 保存图像 #34; 并选择文件格式和 #34;p ng 和 #34;. </li>
	</ol> </li> <li> 报告结果并将反应表位分配给相应的等位基因. </li>
	<li> 将数组结果与 single-antigen 珠子 (审计局) 结果进行比较 (如果可用). 
	注: 使用单位抗原检测抗体反应性, 对固定的 HLA 蛋白进行测试。显示抗体反应性的单个肽属于某些 HLA 等位基因, 如果也包括在审计小组中, 允许直接比较阵列和审计结果。我们以前的研究 17 显示了结果之间的高度相关性, 这表明肽表位显著地促进了审计的整体反应性。此外, 阵列的结果也揭示了氨基酸水平的特异性。
	<ol> <li> 获取来自 HLA 实验室的审计结果, 它提供有关是否和哪些施主等位基因对患者的移植血清反应的信息。步骤5.5 的结果。在阵列分析的基础上还提供有关的同种反应性的相应的供体等位基因阳性信息. </li>
		<li> 比较审计和阵列结果. </li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLAMatchmaker:+a+molecularly+based+algorithm+for+histocompatibility+determination+I.+Description+of+the+algorithm.">HLAMatchmaker: a molecularly based algorithm for histocompatibility determination I. Description of the algorithm.</a> Hum Immunol. 63 (5), 339-352 (2002).</li><li>Duquesnoy, R. J., Marrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLAMatchmaker-based+definition+of+structural+human+leukocyte+antigen+epitopes+detected+by+alloantibodies.">HLAMatchmaker-based definition of structural human leukocyte antigen epitopes detected by alloantibodies.</a> Curr Opin Organ Transplant. 14 (4), 403-409 (2009).</li><li>Wedel, J., Bruneau, S., Kochupurakkal, N., Boneschansker, L., Briscoe, D. 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A., Nickerson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-transplant+assessment+of+donor-reactive,+HLA-specific+antibodies+in+renal+transplantation:+contraindication+vs.+risk.">Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: contraindication vs. risk.</a> Am J Transplant. 3 (12), 1488-1500 (2003).</li><li>Haas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+Banff+schema+for+diagnosis+of+antibody-mediated+rejection+in+renal+allografts.">An updated Banff schema for diagnosis of antibody-mediated rejection in renal allografts.</a> Curr Opin Organ Transplant. 19 (3), 315-322 (2014).</li><li>Cecka, J. M., Reed, E. F., Zachary, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+high-resolution+typing+for+sensitized+patients:+a+solution+in+search+of+a+problem?.">HLA high-resolution typing for sensitized patients: a solution in search of a problem?.</a> Am J Transplant. 15 (4), 855-856 (2015).</li><li>Tambur, A. R., Claas, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+epitopes+as+viewed+by+antibodies:+what+is+it+all+about?.">HLA epitopes as viewed by antibodies: what is it all about?.</a> Am J Transplant. 15 (5), 1148-1154 (2015).</li><li>Robinson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IPD+and+IMGT/HLA+database:+allele+variant+databases.">The IPD and IMGT/HLA database: allele variant databases.</a> Nucleic Acids Res. 43, D423-D431 (2015).</li><li>Gabriel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+typing+by+next-generation+sequencing+-+getting+closer+to+reality.">HLA typing by next-generation sequencing - getting closer to reality.</a> Tissue Antigens. 83 (2), 65-75 (2014).</li><li>Liu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+for+Anti-HLA+Antibody+Detection+Using+Personalized+Peptide+Arrays.">A Novel Method for Anti-HLA Antibody Detection Using Personalized Peptide Arrays.</a> Transplant Direct. 2 (11), (2016).</li><li>Frank, R., Overwin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPOT+synthesis.+Epitope+analysis+with+arrays+of+synthetic+peptides+prepared+on+cellulose+membranes.">SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes.</a> Methods Mol Biol. 66, 149-169 (1996).</li><li>Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SPOT-synthesis+technique.+Synthetic+peptide+arrays+on+membrane+supports--principles+and+applications.">The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports--principles and applications.</a> J Immunol Methods. 267 (1), 13-26 (2002).</li><li>Amartely, H., Iosub-Amir, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+protein-protein+interaction+sites+using+peptide+arrays.">Identifying protein-protein interaction sites using peptide arrays.</a> J Vis Exp. 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S., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+peptide+array+to+identify+binding+motifs+and+interaction+networks+for+modular+domains.">Using peptide array to identify binding motifs and interaction networks for modular domains.</a> Methods Mol Biol. 570, 67-76 (2009).</li><li>Kaczmarek, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-specific+HLA+alloantibodies:+long-term+impact+on+cardiac+allograft+vasculopathy+and+mortality+after+heart+transplant.">Donor-specific HLA alloantibodies: long-term impact on cardiac allograft vasculopathy and mortality after heart transplant.</a> Exp Clin Transplant. 6 (3), 229-235 (2008).</li><li>Claas, F. 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没有利益冲突的声明。

本文所描述的斑点阵列的设计是为了对器官捐献者 HLA 抗原的移植同种特异性进行实验研究。与目前广泛应用于临床的审计方法相比, 抗原阵列法在其灵活设计中具有很大的优势, 能够适应单个捐献者的真实 HLA 序列。新的平台利用快速推进的 DNA 测序技术的潜能, 很快就能够产生准确的 HLA 位序列读数, 而不含糊不清16,27和指定数据库的潜力存储库 HLA 序列...

在世界各地例行进行的器官置换治疗挽救了数百万人的生命。固体器官移植每年在美国约有100患者发生, 而在 waitlists, 由于供应严重短缺, 仍有更多的人接受捐赠器官 (根据该机构提供的资料采购和移植网络-OPTN/UNOS: optn.transplant.hrsa.gov)。器官移植是高度规范的, 以减少器官的浪费和拯救生命, 但用于告知这些规定的科学工具的有效性是有限的。例如, 科学界充分认识到 HLA 分子的高度多态性和使用高分辨率打字和序列类型 (SBT) 的 DNA 的精确基因测试, 这些都是近几年才发展起来的1, 2. 然而, 同种测试方法尚未能够产生各种 HLA 序列作为抗原探针。目前的标准测试使用的是100位抗原的一个恒定的面板, 由 HLA、a、B、C、DQ、DP 和 DR 序列的常见变体组成, 其中3,4,5,6。通常, 实际捐赠者的 HLA 序列不包括在测试小组中, 强迫移植医生和外科医生根据捐赠者的实际序列和相应的 "标准" 的共同的相似性推断供者特定的反应。测试集7,8。因此, 对基于抗体测试结果的拒绝风险进行可靠估计是有挑战性的9,10,11,12。因此, 迫切需要13,14来进行抗体的新的个人自定义测试。
HLA 基因编码的主要组织相容性复合体 (MHC) 受体, 在免疫应答的关键功能6。HLA 基因是已知的最多态基因的人类基因组6。由于 HLA 基因的 DNA 测序策略的快速发展, 新的位变种 (或简称为等位性) 正被发现在一个爆炸性的速率15,16。到 2017年3月, 16755 已证实的等位基因已存入 IMGT/HLA 数据库 (http://www.ebi.ac.uk/ipd/index.html), 其中12351为 I 类和4404类。与此形成鲜明对比的是, 在标准 single-antigen 珠 (审计局) 检测中, 仅有超过100个不同的等位基因被代表, 这通常用于检测器官移植中的抗体。该方法是建立在一个 Luminex 平台上使用流式细胞仪。由于化验采用了一组不变的抗原, 除了小批量到间歇变化在生产中, 抗血清测试可以在个人和实验室之间进行强有力的标准化5。然而, 这项测试无法捕获所有的抗体, 特别是当捐献者的序列不在审计组的时候。虽然根据真实序列定制的捐赠者抗原的生产是可取的, 但在简化必要的生产和测试程序方面仍然存在技术上的挑战。
我们最近在肾脏移植患者的可行性研究中描述了一种替代方法17。该方法采用多肽抗原, 以阵列的形式来探查各科的前后移植血清。每个阵列都是使用点合成方法自定义18,19,20,2122,23 , 生成多肽抗原, 每15氨基酸的长度, 完全基于各自的器官捐献者的 HLA 等位基因 A, B, C, DQA1, DQB1 和 DRB1。用标准的 n-芴甲氧羰基-化学22在纤维素膜上进行斑点合成, 并可与全自动机器人系统19、21并行生成数百个自定义多肽。膜阵列可经受多次剥离和 reprobing 循环。在我们的回顾性研究17中, 我们检测到了在时间序列中收集的存储移植血清 (即移植前后的即) 中抗原模式的变化。本文介绍了该工作流的技术协议, 包括阵列设计、制造、抗血清探测和结果分析。该方法的目的是检测抗体对特定的线性表位移植捐赠人的 HLA 分子。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Peptide array</td>
			<td style="width:141px;">INTAVIS Bioanalytical Instruments</td>
			<td style="width:115px;"></td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Ethanol</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:115px;">E7023</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Ponceau S solution</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:115px;">P7170</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Non-fat milk</td>
			<td style="width:141px;">Bio Rad Laboratories</td>
			<td style="width:115px;">1706404</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">TBST</td>
			<td style="width:141px;">Santa Cruz Biotechnology</td>
			<td style="width:115px;">10711454001</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Goat anti-human IgG&#8211;HRP&#160;</td>
			<td style="width:141px;">ThermoFisher Scientific</td>
			<td style="width:115px;">A18811</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Clarity Western ECL Substrate</td>
			<td style="width:141px;">Bio Rad Laboratories</td>
			<td style="width:115px;">1705061</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Restore Western Blot Stripping Buffer</td>
			<td style="width:141px;">Thermo Scientifics</td>
			<td style="width:115px;">21059</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">ChemiDoc gel imaging system</td>
			<td style="width:141px;">Bio Rad Laboratories</td>
			<td style="width:115px;">1708265&#160;</td>
			<td style="width:155px;"></td>
		</tr>
	</tbody>
</table>

personalized peptide arrays for detection of hla alloantibodies in organ transplantation

在器官移植中, 移植物的功能和寿命关键依赖于控制人白细胞抗原 (HLA) 的免疫排斥反应的成功。组织相容性指导原则是基于抗 hla 免疫的实验室测试, 它要么是预先存在的, 要么是从头生成的 hla 抗体, 构成了主要的移植屏障。目前的测试是建立在一个 single-antigen 珠 (审计) 平台上使用一套固定的〜100预选的重组 HLA 抗原探针移植血清。然而, 在人类中存在着一个更大种类的 hla 类型, 没有两个人, 除了相同的双胞胎谁可以共享相同的 hla 序列组合。虽然先进的 hla 打字和直接测序技术可以准确地捕捉到捐赠者和受体 hla 之间 DNA 序列中的任何不匹配, 但由于其序列表征的多样性, 它无法精确检测抗体特别反对捐献者 HLA 不匹配。我们试图开发一种互补的方法, 使用不同的技术来检测和表征 anti-donor HLA 抗体在个性化的基础上。该筛选工具是一种自定义的肽序列的捐助者 HLA 派生的移植血清的器官受体, 以评估的风险, 抗体介导的拒绝。在一个单一的阵列为一个捐助者-受体对, 多达600独特的肽是根据捐赠人的 HLA 蛋白序列, 每多肽携带至少一个不匹配的残留在 15-氨基酸序列。在我们的实验中比较移植血清对这些阵列的抗原模式, 我们能够检测到抗 HLA 信号的分辨率, 这也使我们能够确定所涉及的免疫表位。这些个性化抗原阵列允许高分辨率检测器官移植中供体特异的 HLA 表位。

在最初的研究中使用数组筛选方法17, 我们总共登记了5肾移植科。我们得到了我们的队列和他们各自的捐助者的 HLA 打字结果。我们也提供了他们的病史和位抗体滴的检查。在我们对这5患者的试验研究中, 我们设计了两种不同的方法: 一个标准数组, 由一个固定的多肽组和个性化的阵列组成, 它们是为每个捐赠者和受体对量身定做的。虽然第一种方法允许我...

Watch this Scientific Journal Video about Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation at JoVE.com

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

器官捐献者与受体对人白细胞抗原 (HLA) 序列的不匹配是器官移植中抗体介导的排斥反应的主要原因。在这里, 我们提出了使用自定义抗原阵列, 根据个别捐赠人的 hla 序列, 以探测 anti-donor hla 抗体在器官接受者。

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

personalized-peptide-arrays-for-detection-hla-alloantibodies-organ

Research

JoVE Journal

Biochemistry

10.0K 次观看.  Northwestern University Feinberg School of Medicine. 器官捐献者与受体对人白细胞抗原 (HLA) 序列的不匹配是器官移植中抗体介导的排斥反应的主要原因。在这里, 我们提出了使用自定义抗原阵列, 根据个别捐赠人的 hla 序列, 以探测 anti-donor hla 抗体在器官接受者。

视频: Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.