Name: detection of human leukocyte antigen biomarkers in breast cancer utilizing label-free biosensor technology
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Description: Watch this Scientific Journal Video about Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology at JoVE.com

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><tbody><tr><td>ResoSens Label-free Bioassay Detection System</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>Avidin Sensitized Bionetic 96-well Plates</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>ResoVu software</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>RL21A Biotinylated TCRm mAb&amp;nbsp;</td><td>PureMHC, LLC</td><td></td><td></td></tr><tr><td>RL9A Biotinylated TCRm mAb&amp;nbsp;</td><td>PureMHC, LLC</td><td></td><td></td></tr><tr><td>FLSL HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: FLSELTQQL</td></tr><tr><td>SLLV HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: SLLMWITQV</td></tr><tr><td>YLEV HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: YLEPGPVTV</td></tr><tr><td>KVL HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: KVLEYVIKV</td></tr><tr><td>Pooled Normal Human Serum&amp;nbsp;</td><td>ThermoFisher</td><td>BP2657100</td><td></td></tr><tr><td>MDA-MB-231 Cell Supernatant</td><td>ATCC</td><td>HTB-26</td><td>Cells grown in IMDM , 10% FBS and 1% penn/strep. Supernatants harvested from 3 day confluent cultures.</td></tr><tr><td>Iscove&#039;s Modification of Dulbecco&#039;s Medium (IMDM)</td><td>Life Technologies</td><td>12440-053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15070-63</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>ThermoFisher</td><td>5318-1</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher</td><td>BP665-1</td><td></td></tr><tr><td>Phosphate Buffered Saline + 0.05% Tween 20 (PBST)</td><td>ThermoFisher</td><td>BP337-500</td><td></td></tr><tr><td>2,2&amp;rsquo;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)</td><td>ThermoFisher</td><td>37615</td><td></td></tr><tr><td>rabbit anti-human &amp;beta;2 microglobulin&amp;nbsp;</td><td>Dako</td><td>A0072</td><td></td></tr><tr><td>Peroxidase conjugated goat anti-rabbit IgG&amp;nbsp;</td><td>Jackson Immunoresearch</td><td>111-035-144</td><td></td></tr><tr><td>Nunc&amp;nbsp; microplates</td><td>ThermoFisher</td><td>439454</td><td></td></tr><tr><td>Synergy 2 microplate reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Immpress Reagent Kit</td><td>Vector</td><td>MP-7402</td><td></td></tr><tr><td>Immpact DAB</td><td>Vector</td><td>SK-4105</td><td></td></tr><tr><td>Hematoxylin QS</td><td>Vector</td><td>H3403</td><td></td></tr><tr><td>IgG2a</td><td>MP BioMedicals</td><td>50328</td><td></td></tr><tr><td>IgG2b</td><td>MP BioMedicals</td><td>50330</td><td></td></tr><tr><td>anti-HLA-A2 (BB7.2)</td><td>Experimmune</td><td></td><td></td></tr><tr><td>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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.9K 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

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening

Here, we present the detailed protocol adopted in the BioMILD screening trial to perform the circulating microRNA signature classifier test for early lung cancer detection.

基于 rna 的液体活检: 血浆 miRNA 特征分类器 (MSC) 在肺癌筛查中的经验

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the ...

科研

癌症研究

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

生物技术环境下的氟黏膜化人乳三糖--用基因编码生物传感器分析

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

生物化学

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

生物工程

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

生物学

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

应用乳腺癌档案材料的多路 RNA 表达方法的优化

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

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 ...