These studies were supported in part by the Development Corporation of Abilene which provides laboratory space and salary support for Experimmune, a Center for Immunotherapeutic Development at Texas Tech University Health Sciences Center and Resonant Sensors Incorporated.

伦理学声明：不标明身份的人体组织和​​血浆样本来自亨德里克医疗中心根据机构审查委员会批准获得协议。 1.加生物素抗体注：该步骤监测是可选的。见备选方案，如果没有监控。 <ol><li>获得市售抗生物素的衍生物涂层板（见材料和设备表 ）。 </li><li>添加50微升磷酸盐缓冲盐水（PBS）pH为7.2至孔中。 注意：预温育的PBS在28℃下，以尽量减少与温度相关的共振位移。 </li><li>开放的生物测定扫描仪软件，然后按照安装向导来定义实验程序，其中包括精选的空白，标准和样品板布局，温度设置（28°C），每分钟的扫描次数（1），与实验长度（ 150分钟，以容纳用于制备步骤）。 </LI&gt; <li>插入制备测定板到标签游离生物测定扫描仪并启动第一扫描。当第一次扫描完成后，选择“自我引用开始”。基线是初始设定在实验开始收集扫描。如果这是在这个特定的盘进行第一读取，允许扫描器运行以平衡温度和消除漂移的基线。参见图2基线之后样品添加的屏幕截图。 </li><li>后基线已经稳定，或至少5扫描，暂停读取和弹出盘。 </li><li>除去PBS倾倒在水槽，并添加50微升RL21A生物素化的抗体在所有，但在板的对照孔[在PBS pH 7.2的10微克/毫升]。添加50微升PBS中至对照孔。 </li><li>板将回到生物测定扫描仪并继续阅读直至饱和，约1.5小时。 </li><li>暂停读取和弹出板。 </li><li>洗<html>板3次，用200μl/孔的磷酸盐缓冲盐水+ 0.05％吐温20（PBST），接着漂洗3次用200μl/孔PBS pH值7.2。重复用PBS。点击多余的PBS从平板上之前，移动到下一个步骤纸巾。 注意：此步骤可以在一个自动洗板机来执行，或者通过倾倒液体成片和用吸管代替PBST洗涤平板3次，用200μlPBST。 </li><li>加入50微升PBS pH值7.2至所有活动的水井。 </li><li>板将回到生物测定扫描仪并继续阅读监视后洗共鸣。 </li><li>停止读取并弹出板。 </li></ol>替代协议第1步： <ol start="13"><li>加在[10微克/毫升的PBS]将50μlRL21A生物素化抗体对所有孔中。 </li><li>在室温下孵育1.5小时或过夜，在4℃。 </li><li>用200μl洗涤平板3次/孔的PBST随后漂洗3次用200μl/孔PBS如在步骤1.9。 </li></ol>jove_title“&gt; 2。加分析物<ol><li>移除PBS并加入每适当分析缓冲液以及50μl的。对于该测定，可商购的正常人血清，使用在PBS中稀释1:20。 </li><li>开放的生物测定扫描仪软件，然后按照安装向导来定义实验过程。 </li><li>插入板进入生物测定扫描仪并开始一个基线读取。如果这是在这个特定的板块进行的第一次读，让扫描仪运行平衡温度，消除漂移基线。 </li><li>后基线已经稳定，或至少5扫描，暂停读取和弹出盘。 </li><li>除去从孔的分析缓冲液。 </li><li>与钉对照的HLA A * 02：01单体轴承相关（FLSL）或不相关的（SLLV，YLEV或KVL）肽浓度范围为[0.625-10微克/毫升]在测定缓冲液。添加50微升标准的板的对照孔</li><li>在测定缓冲液中加入50微升分析物与样品板的孔中。对于该测定法，掺入市售人血清，使用。 </li><li>板将回到生物测定扫描仪并继续阅读直至饱和，约30-60分钟。 </li><li>暂停读取和弹出板。 </li><li>用200μl洗涤平板3次/孔的PBST随后漂洗3次用200μl/孔PBS如在步骤1.9。 </li><li>加入50微升试验缓冲所有活动的水井。 </li><li>板将回到生物测定扫描仪并继续阅读监视后洗共鸣。 </li><li>停止读取并弹出板。 注：测定缓冲液可以是PBS中，组织培养介质，或人血清根据测定的目标在PBS中1:20稀释。 </li></ol> 3.数据分析<ol><li>使用生物测定扫描仪软件或导出原始数据文件到首选统计分析软件进行分析。生物测定仪软件会自动生成基础上，结合曲线实验安装过程中提供的板布局。 注意：如果不使用生物测定扫描仪软件进行分析，按照步骤3.2-3.4。 </li><li>减去基线和阴性对照来自每个随后的数据点。 </li><li>计算平均值和标准偏差为重复孔在每个时间点。 </li><li>生成结合曲线或选择结束点的数据为条形图的曲线图。 </li></ol>

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作者，黛布拉Wawro Weidanz和罗伯特马格努松，分别为行政总裁及首席技术官共振传感器股份有限公司，主要生产在这篇文章中使用的生物测定酶标仪和检测板。

本文所描述的方法引入了对癌症的诊断，这将使在高通量快速检测的病人血清，血浆，尿或唾液样品中的可溶性肽/ HLA复合体96或384孔形式的生物标记物筛选系统。这利用T细胞受体模仿单克隆抗体和一个无标记检测系统的检测系统切分析时间小于1小时相比3.5小时通过常规的ELISA。这种高通量方法使患者对治疗性癌症疫苗或生物药品临床试验的快速分层。目前的方法对这些疾病指标的检测需要个体患者的样品中，既麻烦又费时的过程的亲和纯化和HPLC-MS / MS。虽然这种方法是适合于常规ELISA技术，人们关心的是次要的检测试剂，如抗β2微球蛋白抗体可以改变新生HL的结构一个分子，抑制绑定到TCRM限制检测。无标记检测减轻这些问题。此过程可被修改为在其他无标记系统中使用，如表面等离子体共振系统。然而，相对于在该研究中使用的生物测定系统中，这会大大降低了更高的吞吐量能力。 该协议可以有效地改性的非生物标记的HLA在患者血清和血浆中的检测用粘附到几个关键步骤。初始抗体涂覆过程的监控，建议对于表面的优化，作为&lt;200时移将很可能导致低信号用于随后的检测步骤。如血清蛋白的丰度可能掩盖低浓度分析物的结合的基线和后洗读取都为端点分析是有用的。最后，样品的温度变化可导致井到井变化。建议在所有的样品进行预温育一D中的标签自由biosassay部温度控制器设定在2-3℃的高于室温的温度下进行。冷藏样品应允许足够的时间来达到工作温度。 该协议的未来修改将包括TCRM的板面复。生物测定系统目前适合于井在至少8丛的密度点样。通过引入4-8 TCRM到每个孔中，每个测试样品体积被减少，使每名病人的日益复杂的数据的收集。这种能力可进一步告知患者对于治疗选择。

从细胞内蛋白质剧目的蛋白酶体降解衍生的长度8-11个氨基酸的I类人类白细胞抗原（HLA）的样品的肽和呈现每核细胞1,2的表面上这些肽。与HLA复合体是“自然的生物标志物”，指示每个小区的健康（ 图1），以循环的细胞毒性T淋巴细胞（CTL）。识别相关疾病肽结果消除由CTL违规细胞。因此，适应性免疫应答是消除病毒感染和肿瘤非常有效。然而，免疫系统施加压力，该压力经常促进选择的病毒或肿瘤能够回避有效的CTL反应的。 T细胞受体模仿（TCRM）单克隆抗体（mAb），其识别特定肽/ HLA-A * 02：01配合物，例如那些来自乳腺癌相关蛋白，巨噬细胞迁移抑制因子衍生（MIF19-27）3和NY-ESO-1 157-165，使得能够检测和破坏乳腺癌细胞在缺乏有效的抗肿瘤CTL应答4,5。这代表工作的重点是HLA-A * 0201分子，这是由人口高达30％来表示。然而，识别其他相关的HLA /肽复合物，接着通过生产TCRM使得能够有针对性的免疫疗法和同伴疾病诊断的发展，扩大这项工作的效用，以更广泛的人群。 开往细胞表面上的肽/ HLA复合物的一部分被释放到血浆中。由于肽/ HLA池，现有的方法为挖掘immunopeptidome用于HLA相关联的生物标志物的高度复杂性是费时的。这个过程需要从血浆可溶性HLA的亲和纯化的HLA相关肽通过反相高压液相色谱法分离（RP-HPLC）和肽测序通过串联质谱（MS / MS）6,7。虽然这个过程是一个可行的选择的新的治疗目标的发现，它是一个麻烦的过程作为对HLA相关目标已经在治疗性疫苗或生物制药管道8-10伴侣诊断工具。 TCRM针对这些目标提供了针对分层患者到相关的临床试验，并提供更明智的治疗选择快速伴随诊断的开发工具。 在这项工作中，对于癌症的诊断突破生物标志物筛选系统，提出了利用TCRM和无标记生物测定系统，用于监视以最小的处理步骤的生物相互作用。在无标记生物测定系统是基于一种方法，它利用发生在电介质波导光栅11-14的导模谐振（GMR）效应。当宽带光接触衍射光栅中所需的用于该系统的板，两个特定波长的光被反射。固定化的受体及其配体之间的结合相互作用是实时通过跟踪共振波长偏移用频谱分析仪12监测。独立共振峰发生对入射的TE（电矢量垂直于入射平面）和TM（磁矢量垂直于入射平面）偏振状态提供多个数据点可以潜在地提高检测精度11,14。在该标签的无测定系统使用抗体预致敏板，具有数据分析测定运行时间大约是45分钟。另外，多个患者样品可以询问在96或384孔格式，具有明显的优势比的HPLC-MS / MS基于格式。

<table border="0" cellpadding="0" cellspacing="0" width="1191">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:521px;"><a name="RANGE!A1:D29">Name of the Material/Equipment</a></td>
			<td style="width:195px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ResoSens Label-free Bioassay Detection System</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Avidin Sensitized Bionetic 96-well Plates</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ResoVu software</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RL21A Biotinylated TCRm mAb&#160;</td>
			<td>PureMHC, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RL9A Biotinylated TCRm mAb&#160;</td>
			<td>PureMHC, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FLSL HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: FLSELTQQL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SLLV HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: SLLMWITQV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YLEV HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: YLEPGPVTV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KVL HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: KVLEYVIKV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pooled Normal Human Serum&#160;</td>
			<td>ThermoFisher</td>
			<td>BP2657100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MDA-MB-231 Cell Supernatant</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td style="width:355px;">Cells grown in IMDM , 10% FBS and 1% penn/strep. Supernatants harvested from 3 day confluent cultures.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s Modification of Dulbecco&#39;s Medium (IMDM)</td>
			<td>Life Technologies</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>Life Technologies</td>
			<td>15070-63</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>ThermoFisher</td>
			<td>5318-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>BP665-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline + 0.05% Tween 20 (PBST)</td>
			<td>ThermoFisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8217;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)</td>
			<td>ThermoFisher</td>
			<td>37615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-human &#946;2 microglobulin&#160;</td>
			<td>Dako</td>
			<td>A0072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peroxidase conjugated goat anti-rabbit IgG&#160;</td>
			<td>Jackson Immunoresearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#160; microplates</td>
			<td>ThermoFisher</td>
			<td align="right">439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy 2 microplate reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immpress Reagent Kit</td>
			<td>Vector</td>
			<td>MP-7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immpact DAB</td>
			<td>Vector</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin QS</td>
			<td>Vector</td>
			<td>H3403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a</td>
			<td>MP BioMedicals</td>
			<td>50328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2b</td>
			<td>MP BioMedicals</td>
			<td>50330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-HLA-A2 (BB7.2)</td>
			<td>Experimmune</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 5</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of human leukocyte antigen biomarkers in breast cancer utilizing label-free biosensor technology

据美国癌​​症协会，超过200,000名妇女将被诊断为每年浸润性乳腺癌和大约40,000将死于这种疾病。人类白细胞抗原（HLA）I类样本从细胞蛋白的蛋白酶体降解的肽和通过循环细胞毒性T淋巴细胞（CTL）的呈现在细胞表面上对询问这些片段。的T细胞受体模拟物代（TCRM）单克隆抗体（mAb），其识别乳腺癌特异性肽/ HLA-A * 02：01配合物，例如那些由巨噬细胞迁移抑制因子（MIF 19-27）和NY-ESO-衍生1 157-165能够检测和破坏乳腺癌细胞在缺乏有效的抗肿瘤CTL应答。完整的I类HLA /肽复合物是由乳腺癌细胞脱落，代表潜在相关的癌症生物标志物。在这项工作中，对于癌症的诊断突破生物标志物筛选系统š掺入T细胞受体模拟物的单克隆抗体结合使用导模谐振（GMR）传感器技术的新颖，无标记生物传感器呈现。检测棚-MIF / HLA-A *的02：01配合在MDA-MB-231细胞上清液，掺入人血清，和患者血浆是证明。这项工作的影响，可以通过发展配套病诊断有针对性的免疫疗法彻底改变个性化的药。

01分子3：使用充分表征的TCRM，RL21A，一个鼠IgG2a单克隆抗体，其特异性识别的肽（MIF 19-27或FLSL）的HLA-A * 02的上下文中进行的代表性组实验。同源肽/ HLA单体，以及不相关的HLA单体15，被用来证明所述的过程。 图3示出了测定形式。 RL21A是特异于FLSL / HLA单体很少的交叉反应性与其他的肽/ HLA复合物3。这种特殊性是使用无标记的生物测定系统（ 图4）概括。该实验的标签游离生物测定系统的灵敏度是在低纳摩尔范围如通过在固定化FLSL / HLA单体（ 图5）的RL21A抗 ​​体的滴定法测定。虽然背景信号的血清样品中显著增加，Specific检测尖刺人血清FLSL / HLA单体是在图6中实现的，并具有浓度梯度是容易辨别这些样品中。最后，来自MDA-MB-231细胞系的上清液，先前表明呈现FLSL / HLA-A * 02：01的分子，示出了使用这种标签的分类测定平台（ 图7），以含有可溶性FLSL单体。随后加入FLSL / HLA单体的增加（ 图7）上RL21A信号。由于该系统的灵敏度，以及向井变化包括观察如在图4和5的标准偏差的函数，则会发生由于温度波动而导致的负谐振移位。这些变化可以显着地由预孵育所有样品和所述生物测定读板器在稍过量的室温（ 即 28℃）降低的温度。 在图8中 ，简单地说，96- 嗯聚苯乙烯板用[10微克/毫升] RL21A的2小时，在室温下，用PBST洗涤，阻断用0.5％的低脂肪牛奶，用PBST洗涤，并在室温下用连续稀释的培养2小时FLSL的HLA单体在正常人血清1:10稀释在PBS中，以产生标准曲线或患者血浆在PBS中1:10稀释。将板用PBST洗涤，孵育1小时，在室温下，用兔抗人β2微球蛋白[1：5000〕，以检测完整的HLA，并在PBST中洗涤。然后将板孵育30分钟，用山羊抗兔IgG [1：10,000]和用PBST洗涤。使用ABTS（2,2&#39;- Azinobis [3-乙基苯并噻唑-6-磺酸] -diammonium盐）基片用15分钟温育时间，并在上一酶标仪405nm处观察到的，进行比色检测。 FLSL / HLA在三个病人的样品进行检测。 在图8B中 ，患者血浆RL.064物以1:20稀释在PBS中，然后添加编到板和无标记检测器上60分钟监测。具体检测的病人血清FLSL / HLA复合物来实现的。利用图表和统计软件（P &lt;0.05）进行t检验。 在图8C中 ，组织切片进行染色，在1μg/ ml的小鼠抗HLA-A2（BB7.2）作为阳性对照，RL21A，的IgG2b和IgG2a分别作为阴性对照。使用抗小鼠检测试剂盒，DAB（diaminobenzadine）和苏木QS对核染色的指示由生产染色进行检测。通过RL21A肿瘤组织染色证实呈现FLSL / HLA复合物。 <img alt="图1" src="/files/ftp_upload/52159/52159fig1highres.jpg" /> 图1：肿瘤抗原呈递由I类人类白细胞抗原癌性变换是一种细胞内紊乱。 HLA样本intracellulAR蛋白和揭示在细胞表面与癌症相关的变化。 CTL和TCRM能够通过的HLA肽复合物独特的那些细胞识别癌细胞。 <img alt="图2" src="/files/ftp_upload/52159/52159fig2highres.jpg" /> 图2：示出了初始的基准数据采集中的生物测定扫描仪软件的数据采集的截图示例扫描随后RL21A抗 ​​体除涂有FLSL / HLA复合物为10μg/ ml的板。抗体的浓度表示。每一行代表一个单独的监控以及随着时间的推移。 <img alt="图3" src="/files/ftp_upload/52159/52159fig3highres.jpg" /> 图3：试验格式插图抗体固定在测定板捕获相对的衍射光栅表面埃文特肽/溶液中HLA复合体。 <img alt="图4" src="/files/ftp_upload/52159/52159fig4highres.jpg" /> 图4：特异性RL21A的FLSL / HLA复杂的示范生物素RL21A TCRM的特异性及其相关（FLSL）HLA单体相比无关HLA单体（YLEV，SLLV和KVL）的。生物素化的RL21A TCRM [10微克/毫升]的固定化的抗生物素蛋白涂覆的试验板表面上，并利用未标记的相关或不相关的HLA单体[10微克/毫升]在PBS中进行检测。 RL21A是特异于FLSL / HLA复合。双向ANOVA，使用绘图和统计软件（P &lt;0.001）进行。 <img alt="图5" src="/files/ftp_upload/52159/52159fig5highres.jpg" /> 图5：结合RL21A敏感性与其同源肽/ HLA复合插图的检测系统，效的对限制和约束RL21A的敏感性，FLSL / HLA复杂。生物素化的HLA单体FLSL [10微克/毫升]被固定在测定板表面上的和未标记RL21A加入到该板在PBS中连续稀释所指示的。检测该系统是在低纳摩尔范围。 <img alt="图6" src="/files/ftp_upload/52159/52159fig6highres.jpg" /> 图6：在尖刺人血清示范HLA在人血清中检测的检测的HLA配合物 。生物素化的RL21A TCRM [10微克/毫升]的固定化的抗生物素蛋白包被测定板表面上。合并的正常人血清掺有相关（FLSL）或不相关的（SLLV）的HLA单体，以1:20稀释在PBS中，然后添加至板，并在无标记检测器上60分钟监测。特异性检测人血清中FLSL / HLA复合物来实现的。在一般情况下，背景信号（不相干SLLV单体）是高呃用于稀释的血清样品相比，在PBS中纯化的分析物（参见图4）。双向ANOVA，使用绘图和统计软件（P &lt;0.0001）中进行。 <img alt="图7" src="/files/ftp_upload/52159/52159fig7highres.jpg" /> 图7：在乳腺癌细胞培养物上清液中检测到可溶性HLA复合物来检测的HLA乳腺癌细胞上清液的能力证实。生物素化的RL21A TCRM，RL9A TCRM（阴性对照），和同种型对照被固定在测定板表面上。掺入[5微克/毫升]和无稀释剂的MDA-231细胞培养上清中加入并结合被监测的无标记检测器上60分钟。 FLSL和SLLV HLA单体飙升样本代表了RL9A和RL21A结合阳性对照。单向ANOVA，使用绘图和统计软件（P &lt;0.0001）中进行。 图8：特异性检测患者样品中FLSL / HLA复合物（A）传统的酶联免疫吸附试验（ELISA）是用来检测FLSL / HLA复合物的特定病人的血浆。 （B）中的生物素化RL21A TCRM或IgG2a的[10微克/毫升]被固定在抗生物素蛋白测定板。如前所述3验证FLSL / HLA复合物的肿瘤呈现（C）从病人RL.064乳腺肿瘤组织染色。 <a href="https://www.jove.com/files/ftp_upload/52159/52159fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology at JoVE.com

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

完整的I类HLA /肽复合物是由肿瘤细胞脱落，代表了潜在的癌症相关的生物标志物。利用无标记传感器技术和T细胞受体的模仿的单克隆抗体，检测棚-MIF / HLA-A *的02：01配合在MDA-MB-231细胞上清液，掺入人血清，并且患者血浆证明，有利的发展一种新型癌症诊断平​​台。

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

detection-human-leukocyte-antigen-biomarkers-breast-cancer-utilizing

Research

JoVE Journal

Immunology and Infection

13.4K 次观看.  Texas Tech University Health Sciences Center. 完整的I类HLA /肽复合物是由肿瘤细胞脱落，代表了潜在的癌症相关的生物标志物。利用无标记传感器技术和T细胞受体的模仿的单克隆抗体，检测棚-MIF / HLA-A *的02：01配合在MDA-MB-231细胞上清液，掺入人血清，并且患者血浆证明，有利的发展一种新型癌症诊断平​​台。 

视频: Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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 Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

通过横向流技术检测在血液系统恶性肿瘤患者侵袭性肺曲菌病

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...