研究提出支持的查尔斯河。

1。的MFIA程序的说明（图1） <ol><li>在的MFIA需要Charles River的抗原包被聚苯乙烯微球（珠），测试血清标记试剂（BAG，SPE）和缓冲区（小学稀释液，测定缓冲液）。 </li><li>试剂逐步加入到96孔过滤底微量滴定板的孔中。 </li><li>该MFIA是异构测试含义孵育随后由过滤器的洗涤步骤，以除去未结合的血清成分，或标记试剂进行。洗涤溶液添加到板的孔，以及过滤器的底部，保留珠粒通过抽吸除去。 </li><li> MFIA检测在室温下（27°C + / -2°C）。 </li><li>检测到抗原 - 抗体复合物在测试过程中血清孵育步骤形成与生物素化的山羊抗物种结合物（袋），其次，由R-藻红蛋白标记的链霉亲和素（SPE）的孵育。 </li><li>以测定读取器，珠粒通过一个在同一时间通过。检测器，在那里，它们暴露于两个激光器。一个激光激励内部识别磁珠的颜色集合，这对应于一个试验的染料;其他激发的藻红蛋白的测定期间捕获的报告染料。预定数目的珠，每次测定读取，作为中位数荧光指数（MFI）和藻红蛋白的荧光强度的报告。 <img alt="图1。" src="/files/ftp_upload/3715/3715fig1.jpg" /> 图1。MFIA程序。的xMAP基于MFIA是悬浮芯片采用彩色编码的聚苯乙烯5.6微米的珠抗原（或控制）的共价连接。由于珠粒在100个不同的颜色集，多达100个不同的检测可以在单井含量步骤执行，在过滤器的底微量滴定板中进行洗涤，使珠可以通过抽吸的真空歧管上。反应是阅读与Luminex公司的xMAP 100荧光。的强度phycoerythrin荧光报告为平均荧光指数（MFI） </li></ol> 2。在开始前，请注意以下几点: <ol><li> MFIA珠是光敏感<ol><li>直接光线照射量的限制是至关重要的。例行试验过程中发生的正常曝光量是可以接受的，但长期接触可导致漂白的珠子，这使他们无法读取的含量读者。 </li><li>的MFIA珠总是涡旋成悬浮，然后超声处理（10-30秒）在使用前，这是至关重要的成功的测定。 </li><li>从单珠含量读者收集信息。聚合珠群会导致更长的读取时间。 </li></ol></li></ol><ol start="2"><li>一定要保持测试板的板盖覆盖在所有孵化的步骤，以避免蒸发的分析解决方案。 </li><li>个人防护装备:实验室外套，GL在任何时候，在实验室环境中工作时，应佩戴oves和保护眼睛的。 </li></ol> 3。试剂<ol><li>表1列出了大部分所需的材料，以建立一个内部MFIA的实验室。附加的或重复的项目可能需要根据您的具体需求。请注意，这个清单不包括从查尔斯河（MFIA珠，控制和补充试剂）试剂购买。生物素化的结合物与链霉亲和标签的藻红蛋白的几个商业来源是可用的。但是从Charles River试剂滴定，以产生最佳的信号噪声的得分与我们的试剂，使用替代供应商或试剂等，不推荐。 表1中 。 CR-RADS使用一个BioPlex的的的的悬浮阵列从BioRad公司的阅读器和自动洗板机从BioTek的。等效的仪器，从不同的供应商提供。 <table border="1"><tboDY&gt; <tr><td> 项目 </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td> 设备 </td><td></td><td></td></tr><tr><td>悬浮芯片阅读器</td><td> * BioRad公司</td><td> 171-000205 </td></tr><tr><td> 96孔微孔板垫圈</td><td>百特</td><td> ELX50/8FMW </td></tr><tr><td>超声波清洗机/浴</td><td>科尔帕尔默</td><td> EW0884900 </td></tr><tr><td>模拟旋涡混合器</td><td> VWR </td><td> 58816-121 </td></tr><tr><td> -20°C冰箱</td><td>各个</td><td></td></tr><tr><td> 4°C冰箱</td><td>各个</td><td></td></tr><tr><td> 12道移液器20-200μl的</td><td> VWR </td><td> 83009-718 </td></tr><tr><td>轨道板振动筛</td><td> VWR /实验室线</td><td> 57019-600 </td></tr><tr><td>单道移液器，20-200μl的搭配秘诀</td><td> VWR </td><td> 83009-732 </td></tr><tr><td>单道移液器，2-20μL，搭配秘诀</td><td> VWR </td><td> 83009-726 </td></tr><tr><td>单道移液器，1001000μL，搭配秘诀</td><td> VWR </td><td> 83009-736 </td></tr><tr><td> Vacushield通风装置</td><td> VWR </td><td> 55095-006 </td></tr><tr><td>真空压力泵</td><td> VWR </td><td> 54908-037 </td></tr><tr><td>真空系统废液池</td><td> VWR </td><td> 80200-640 </td></tr><tr><td> 耗材 </td><td></td><td></td></tr><tr><td> 96 polystryrene板</td><td> Fisher Scientific则</td><td> 14-245-145 </td></tr><tr><td> MultiScreen的HTS-BV板，1.2微米过滤器，苯乙烯</td><td>密理博</td><td> MSBV N12 50 </td></tr><tr><td> 15ml锥形管（聚丙烯） </td><td>萨尔斯塔特</td><td> 62554.002 </td></tr><tr><td> 50ml锥形管（聚丙烯） </td><td>萨尔斯塔特</td><td> 62547.004 </td></tr><tr><td>血清瓶</td><td>萨尔斯塔特</td><td> 72694.007 </td></tr><tr><td>铝箔</td><td> VWR </td><td> 89079-068 </td></tr><tr><td> 1L瓶，无菌</td><td> VWR </td><td> 28199-246 </td></tr><tr><td> 0.22μm的瓶顶过滤器</td><td> VWR </td><td> 28199-307 </td></tr><tr><td>试剂水库（100毫升） </td><td> VWR </td><td> 82026-356 </td></tr><tr><td> 5ML，10ML，25ML移液管分配液体试剂</td><td> VWR </td><td></td></tr><tr><td> 试剂 </td><td></td><td></td></tr><tr><td> PBS，pH值7.4 1 BSA，粉（袋） </td><td>西格玛</td><td> P3813 </td></tr><tr><td>的Proclin 300 </td><td> SIGMA / Supelco公司</td><td> 48912-U </td></tr><tr><td>胶版纸球</td><td> Luminex公司</td><td>不同类型</td></tr></tbody></table></li></ol> 4。试剂准备<ol><li>需要两个缓冲区为MFIA程序。主要稀释剂是由Charles River提供，并包含一种特有的阻断剂，以减少非特异性的蛋白质的相互作用。在您的设备与市售试剂检测洗涤液。 </li><li>含量洗涤缓冲液:PBS / 1％BSA的pH为7.4作为从Sigma-Aldrich公司的粉末是可用的。 <ol><li>通过过滤的微粒的去除是至关重要的，因为这些颗粒可导致木屐在测定阅读器。一个0.2micron到无菌的，贴有标签的容器瓶顶过滤器，过滤器的缓冲。 </li><li> BAG和SPE稀释至含量浓度的洗涤液，2X工作。注:SPE是光敏感和sh·乌尔德·有限的光照射。 </li></ol></li></ol> 5。样品制备<ol><li>装配试验材料，试剂，一次性的。 <ol><li>多和单通道微量移液器和技巧</li><li> 96 - 孔低蛋白结合的微量滴定板（血清稀释）和过滤器的底板（MFIA测定） </li><li>根据标准程序采集血液样本。允许的血液标本血块完全由它们在室温下保持至少30分钟，然后离心和血清的去除。应振荡Undilute血清，简单地混合血清成分采样前。 </li><li>最终测试为MFIA稀释为1/50。这是必要的，使测定了2倍（1/25）样品的测试血清。未稀释的试验血清加1份血清，24份MFIA主要稀释剂稀释。测试血清中的96孔微量滴定板中的组织极大地降低了传输时间对样品的工商业污水附加费T形钢板，强烈推荐。 </li></ol></li></ol> 6。试板准备<ol><li>预润湿的96孔测试板，是必需的，以保证正确，即使以及疏散。即使整个板块将不会被使用需要这一步最初，你不会有，添加后续试剂或洗涤缓冲液井空。 <ol><li>一些实验室运行不到一个完整的96孔板的密封关闭未使用的测试板，并计划在以后的日子重新使用这些井的井。我们不建议使用此方法，因为它可能会影响测试板的适当的过滤。 </li></ol></li></ol><ol start="2"><li>正确的测试板愿望的MFIA试验的成功是至关重要的。疏散样品井约5-10秒。吸气阱内容太快可导致胎圈聚集和缓慢的读取时间结束时的测定。 </li><li>后，每瓦特的试验板用纸巾吸干底面火山灰的步骤，以确保液体的排水已停止。如果涂抹试验板，可以导致液体试剂，毛细作用的井孵化过程和结果，在失败的山羊抗物种和/或种属的IgG珠分数。这些珠被设计得分MFI内的预先确定的范围内时，适量的主血清和标记试剂被加入到各孔。 </li></ol> 7。 MFIA珠混悬剂<ol><li>这是至关重要珠粒总是涡旋和超声处理，在使用前的成功的测定。收集信息，分析读者从单珠，珠聚集体或团块会导致更长的读取时间。 </li><li>涡的股票加上珠悬浮液（通常为10±5秒） </li><li>重悬珠其次超声波仪，浴中通过超声处理10-20秒。 </li><li>稀释20倍的股票停牌珠悬浮在主稀释2倍的工作。免除的2倍珠悬浮液50μL每个检测的预湿试验板。 </li></ol> 8。此外，试验和对照血清的测试板<ol><li>移取50μl的每一个2X测试和控制血清测试根据预定义的板图板。安全的盖子，板与弹性带或铝箔孵育轨道摇床，在黑暗中，在室温下，持续60分钟的试验板。 <ol><li>摇床转速应大于400，但低于700RPM。这是至关重要的，珠粒被保持在悬浮液中，以允许访问所有表面耦合抗原血清抗体。否则保持暂停珠粒将导致低IgG珠控制分数，并测定将需要被重复。 </li></ol></li></ol> 9。洗衣机的测试板<ol><li>使用真空歧管吸出很好的内容。疏散样品井约5-10秒。 </li><li>吸干佩普试验板的底面与ŕ后，每毛巾，以确保排液已停止。 </li><li>洗涤液的含量在50μl重悬珠。珠粒不能干出在测定过程中，这一点很重要。 </li></ol> 10。 BAG和SPE的测试板<ol><li>分液50μL2X工作液袋的所有MFIA珠孔中。的盖子固定到板与弹性带或铝箔。如上述，在黑暗中，在室温下振荡孵育，持续30分钟的试验板。 <ol><li>保温后洗的测试板和血清此外如前所述悬浮含量洗涤液珠。 </li></ol></li></ol><ol start="2"><li>分配50μL的2倍稀释至所有的孔中MFIA珠的SPE。的盖子固定到板与弹性带或铝箔。如上述，在黑暗中，在室温下振荡孵育，持续30分钟的试验板。 <ol><li>在此之后INCU的cDNA克隆洗的测试板和悬浮磁珠125μl洗涤液的含量和摇板〜两分钟之前到检测器悬浮珠。 </li></ol></li></ol> 11。阅读测试板<ol><li>试验板放置到测定读取器，在10分钟内再悬浮珠粒。在电源故障或其他事件的事件，测试板可以被存储在黑暗中在室温下，时间长达12小时，直到准备好读。 <ol><li>观察第一个样本，以验证所选择的珠面板相匹配的配置文件中的测试井珠。如果有是属于协议显示他们的指定的"胎圈区域&#39;珠，选择一个不正确的更新已经取得进展。 </li><li>如果小珠已经正确地悬浮，应填写在其指定的"区域"在分析阅读软件表示。 </li><li>如果小珠聚集在他们的地区，他们将填补一个缓慢的率。您可以删除测试版的读者和手动悬浮井。捣碎各孔与多通道移液器的3-4倍，以协助在分手胎圈粒料。更换测试板插入读卡器，然后继续。 </li></ol></li></ol> 12。代表性的成果<ol><li>最低数量的珠子，读法和藻红蛋白荧光强度的报道为平均荧光指数（MFI），范围从0到32,667。在比较计数25，50或100，每孔珠，我们看到无显着性差异，并定期计算每剂25珠，每个样品。检查结果报告的错误，如的珠数量不足或未能抗体/抗IgG珠分数。这些珠被设计为"得分"内的预先确定的MFI范围时，适量的主血清和标记试剂被加入到各孔中。样品井的错误，（珠计数低），或者未IgG​​对照珠分数重复进行，以获得有效的结果。 </li><li>结果导出到Excel工作表中。对于我们的数据解释分析分配: <ol><li>甲组织控制（TC）测试:对于大多数的啮齿动物MFIA检测，组织控制是野生型杆状病毒感染的昆虫细胞的提取物。 </li><li>一种含量托夫:此值是最小荧光预期调整为每个检测，以最大限度地提高诊断的准确性。对于大多数检测，荧光截止为3000。 </li></ol></li></ol><ol start="3"><li>净的中位数荧光指数（MFI）来计算由下面的公式为每个测定（组织和IgG对照测试除外）。换句话说，具体的测定信号的总测定的MFI减去这个值再除以1000的TC（背景）的MFI 0-32代替0-32,667之间简单地设置的范围内的净MFI。 净MFI =（MFI 抗体检测 - MFI 组织控制 ）/ 1000 氖吨MFI和TC MFI被转换成分数相比，针对每种抗原的含量截止。 <img alt="图2。" src="/files/ftp_upload/3715/3715fig2.jpg" /> 图2。的代表MFIA数据抗体测试血清与所有控制传递。 </li></ol><ol start="4"><li> 12.4 MFIA解释<ol><li>试验板的结果只应被解释，如果系统的适宜控制和血清控制（IgG抗体/抗-IgG珠）的结果满足认可准则，表2中列出。这两个IgG对照珠失败可能表明了一些程序上的错误，如不适当稀释或不足量的共轭或SPE，不正确的测试血清稀释或使用的血清从一个immunocomprimised的动物。 表2 <table border="1"><tbody><tr><td colspan="3"> 验收标准含量控制 </td></tr><tr><td rowspan="2"> 控制 </td&gt; <td colspan="2"> 可以接受的结果 </td></tr><tr><td> 得分 </td><td> 分类 </td></tr><tr><td>高范围免疫血清</td><td> ≥4.5 </td><td>积极</td></tr><tr><td>低范围免疫血清</td><td> ≥1.5 </td><td> ≥边缘</td></tr><tr><td>非免疫动物血清</td><td> &lt;2.5 </td><td> ≤边缘</td></tr><tr><td>稀释液</td><td> &lt;2.5 </td><td> ≤边缘</td></tr><tr><td>免疫球蛋白订珠* </td><td> ≥Cutoff/1000 </td><td>通过</td></tr></tbody></table> *珠涂与种属特异性抗检测血清免疫球蛋白（Ig）设置:此示例适宜控制分数没有增加样本量不足，可能会导致过高的样品稀释，不正确的物种或检测血清的免疫浩ST。 </li></ol><ol start="2"><li>如果有任何的测试板含量控制除了高频范围内的血清免疫失败，结果是不能接受的，必须重复进行。通过低范围血清控​​制分数线是至关重要的，因为它演示了检测的灵敏度水平测试板。 </li><li>的山羊抗-IgG抗体珠种得分是基于主要的测试血清的量加入到测定。如果这个控制胎圈失败，但种的IgG抗体通过（井B1，图3），则它表明足够的检测试剂（BAG，SPE）以及被添加到测试，它是一​​种血清相关的问题。如果测试试剂的量是不足的，在一个特定的井的IgG和抗-IgG控制珠将失败（E1，图3）。 <img alt="图3。" src="/files/ftp_upload/3715/3715fig3.jpg" /> 图3。的代表MFIA测试结果与故障（橙色）。井A1，C1和F1表明，TC（组织无功）失败TC得分表示在括号中。韦尔斯B1和E1示出了两种类型的IgG抗体的内部控制，故障，受试抗体不足，（B1）或测试试剂（E1）袋/ SPE分别。 </li></ol><ol start="4"><li>如果测试板含量控制的效果是令人满意的，单独的检测分数归类为示于表3。这是非常重要的替代测试方法，以确认任何边缘或积极的发现。 表3 <table border="1"><tbody><tr><td colspan="4"> MFIA分数分类 </td></tr><tr><td colspan="3"> 得分 </td><td rowspan="2"> 分类 </td></tr><tr><td> TC </td><td> 净 </td><td> TC +网* </td></tr><tr><td rowspan="2"> ≥2 </td><td> &lt;0.5 </td><td> &lt;2.5 </td><td>负极（ - ） </td></tr><tr><td&gt;≥0.5 </td><td> ≥2.5 </td><td> TC反应（T） </td></tr><tr><td rowspan="3"> &lt;2 </td><td> &lt;1.5 </td><td></td><td> - </td></tr><tr><td> 1.5≤X &lt;2.5 </td><td></td><td>边缘（B） </td></tr><tr><td> ≥2.5 </td><td></td><td>正（+） </td></tr></tbody></table> *分类负仍然可以被确定的一个非零的TC得分提供的TC +净得分（=得分）银是负</li></ol></li></ol>

<ol>
	<li>Jacobson, R. H., 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=Principles+of+validation+of+diagnostic+assays+for+infectious+diseases.">Principles of validation of diagnostic assays for infectious diseases.</a> Manual of Standards for Diagnostic Tests and Vaccines. , 8-15 (1996).</li><li>Wright, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+standards+for+test+methods+and+reference+sera+for+diagnostic+tests+for+antibody+detection.">International standards for test methods and reference sera for diagnostic tests for antibody detection.</a> Rev. sci. tech. Off. int. Epiz. 17 (2), 527-533 (1998).</li><li>Pepe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Statistical Evaluation of Medical Tests for Classification and Prediction. , (2003).</li><li>Barr, M. 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=Comparison+and+interpretation+of+diagnostic+tests+for+feline+immunodeficiency+virus+infection.">Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.</a> J. Am. Vet. Med. Assoc. 199, 1377-1381 (1991).</li></ol>

所有的作者查尔斯河RADS工作，动物血清学检测服务和试剂的主要供应商。

MFIA测试过程中是高效率，需要更少的设备和更小的体积比传统的单重测试样品和试剂。的多路传输系统的功能，为用户提供了灵活性，屏幕上同时多株或血清型的实验室啮齿动物（例如冠状病毒，细小病毒等）的共同代理。这也使得我们，感兴趣的区域（即特定的病毒家族）的基础上设计定制的胎圈板，是适应其他类型的生物分子，包括细胞因子和其他生物标志物筛选。此外，它允许我们将内部控制的实验来验证样品与系统适用性，从而保证结果的准确性。这些措施包括组织控制和IgG抗体检验血清种免疫球蛋白（αIg）的镀膜珠集，以评估样品的适用性。一个组织控制磁珠检测的非特异性结合的血清免疫球蛋白和αIg珠控制确认该血清已被添加并含有足够的免疫球蛋白浓度。种免疫球蛋白血清，另一个控制珠，涂表明，标记的试剂和检测器的正常工作。其他市售的多重格式的血清学检测（即芯片，ImmunoComb）可能无法提供同一级别的结果确认。 的能力，缩小的可能来源分析失败之前重新测试可以帮助研究者节省时间和材料。关键方面的MFIA的，应确认之前重复不合格的样本。由于在环境温度下进行测定，应验证，实验室的温度是约27℃±2℃，较高的温度可导致比对照和样品的预期分数降低。洗衣机也许是最关键的一步检测。保险试验板正确洗涤，吸干，并再悬浮可以消除由于低胎圈计数（由于凝集珠）或添加试剂不足（丢失通过排汗满分试验板过滤器底部）的抽样误差绝大多数。我们经常在数法（代理人）25珠，并没有发现任何统计学上的差异，结果计数较高的珠子，这也将导致一个较长的读取时间为板。 我们已经进行了全面的验证研究MFIA数种，常用的实验动物（小鼠，大鼠，仓鼠，豚鼠和兔）证明诊断的准确度，重复性和耐用性测试大量已知阳性和阴性血清样品，并比较其ELISA，IFA和MFIA结果的。的检出限（即，标准血清免疫滴定终点）MFIA相媲美，而且在某些情况下，超越，那些相应的ELISA。测量SPF啮齿类动物血清，诊断的特异性，超过99％的整体通信betweeŇELISA和MFIA已知的阳性和已知的阴性血清进行大于95％。总之，这些结果证明，多路MFIA是一个很好的替代单一ELISA，是适合其用途，即在常规的血清学监测实验室动物的殖民地。

multiplexed fluorometric immunoassay testing methodology and troubleshooting

为了确保质量，在生物医学研究中使用的动物模型，我们开发了大量的诊断测试策略和方法来确定，如果动物被暴露在偶然的传染性病原体（如病毒，支原体和其他挑剔的微生物）。感染免疫功能的动物通常是短暂的，但常常可以检测到数天至数周之内血清抗体反应，感染和坚持的整个生命周期的主机。血清学检查是主要的诊断方法，实验室动物进行监控。间接酶联免疫吸附试验（ELISA）在历史上一直是主要的筛选方法为血清学监测。作为单重进行的ELISA，其中微生物的抗原 - 抗体反应测定，每。比较MFIA进行作为一个复用的测定。由于微球有100个不同的颜色集，可以进行多达100种不同的检测simultaneously在一个单一的微孔板。这种创新降低血清，试剂和一次性用品的例行测试，同时增加的量，获得的信息以及从一个单一的测试所需的量。此外，我们还可以将多个内部控制珠，以确认样品与系统适用性，从而保证结果的准确性。这些措施包括组织控制和IgG抗体检验血清种免疫球蛋白（αIg）的镀膜珠集，以评估样品的适用性。在ELISA和IFA，组织控制检测到的非特异性结合的血清免疫球蛋白。 （血清对照）αIg控制证实，血清已被添加并包含足够的IgG控制系统适应性控制（珠），涂有血清种免疫球蛋白，免疫球蛋白浓度，同时表明，标记试剂和Luminex阅读器是否正常工作。

Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

使用Luminex公司的xMAP微球技术，我们开发了复用荧光免疫法（MFIA）为血清学监测各种实验室动物物种的。 MFIA是抗原，组织控制或免疫球蛋白共价连接到颜色编码的聚苯乙烯微球的悬浮液微阵列。解决MFIA测试方法以及各种疑难排解主题。

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

multiplexed-fluorometric-immunoassay-testing-methodology

Research

JoVE Journal

Biology

28.3K 次观看.  Charles River. 使用Luminex公司的xMAP微球技术，我们开发了复用荧光免疫法（MFIA）为血清学监测各种实验室动物物种的。 MFIA是抗原，组织控制或免疫球蛋白共价连接到颜色编码的聚苯乙烯微球的悬浮液微阵列。解决MFIA测试方法以及各种疑难排解主题。

视频: Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

科研

免疫与感染

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

癌症研究

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

手动复用荧光免疫学的优化、设计和避免陷阱

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

癌症组织中免疫细胞标志物的原位免疫组织化学检测

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

作者聚焦：使用荧光免疫组织化学高效检测癌症组织中的免疫细胞浸润

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

多重环状荧光免疫组化

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

复用荧光免疫检测方法和故障排除

摘要

探索更多视频

此视频中的章节

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

手动复用荧光免疫学的优化、设计和避免陷阱

多重环状荧光免疫组化

多重环状荧光免疫组化

多重环状荧光免疫组化

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别