Name: developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens
Uploaded: 2016-09-12T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens at JoVE.com

Clarissa Curioso was supported through an appointment to the Research Participation Program at the U.S. Environmental Protection Agency administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA.

从机构审查委员会获得批准（IRB＃08-1844，北卡罗莱纳州教堂山分校，北卡罗来纳大学，美国），用于刺激沟唾液样本从博克龙海滩，波多黎各，前往沙滩的集合作为美国的一部分环保局（USEPA）国家流行病学和娱乐（NEEAR）水研究23环境评估，评估中游泳暴露和疾病。研究对象签署知情同意书，并指导对训练的USEPA承包商使用唾液收集装置。唾液样品运送在冰上，在接收，它们被离心，并按照所描述4在-80℃下储存。 1.珠激活<ol><li>通过涡旋和超声处理20秒和存量珠（400微升）到微量离心管转移约5.0×10 6个悬浮珠集的股票。 注:t他珠以12.5×10 6珠/ ml的浓度被提供。 </li><li>通过以10,000 xg离心离心2分钟沉淀库存珠。 </li><li>涡旋和超声处理20秒弃上清，100微升重悬珠包衣蒸馏水（DH 2 O）。重复步骤1.2。 </li><li>除去上清液并通过涡旋和超声处理20秒重悬洗涤珠在80μl的100mM磷酸二氢钠，pH值6.2的。 </li><li>在使用前，立即加入200微升的dh 2 O至10毫克硫代NHS等分使50毫克/毫升N- -hydroxysulfosuccinimide（硫代NHS）的解决方案。涡旋混合。 </li><li>添加10微升50毫克/毫升硫代NHS的至珠。涡旋混合。 </li><li>使用前立即，使50毫克/毫升1-乙基- [3-二甲氨基丙基]碳二亚胺盐酸盐（EDC）溶液中加入200μl的蒸馏水（DH 2 O），以10毫克EDC等分试样。涡旋混合。 </li><li>加入10微升50毫克/毫升的EDC溶液到珠子。涡旋混合。孵育小珠在室温下20分钟，在黑暗中，在10分钟的间隔通过涡旋混合。沉淀通过微量离心活化珠以10,000 xg离心2分钟。 </li><li>除去上清液并通过涡旋和超声处理20秒重悬珠子在250微升50毫2- [N-吗啉代]乙磺酸（MES），pH值5.0。 </li><li>沉淀通过微量离心活化珠以10,000 xg离心2分钟。 </li><li>重复步骤1.9和1.10。 注意:这提供了总共两个洗涤用50mM MES，pH值5.0。 </li><li>悬浮通过涡旋和超声处理在100μl50mM的MES，pH 5.0的珠为20秒。 </li></ol> 2.珠联轴器<ol><li>耦合抗原使用表1所示的浓度的珠集。 </li><li>每种抗原添加至活化的珠子和使总体积为500微升的50mM MES，pH值5.0。混合抗原的ð珠涡旋。 </li><li>孵育2小时的抗原和珠在室温下在黑暗中由旋转（〜15转）混合。沉淀通过微量离心将偶联珠以10,000 xg离心2分钟。 </li><li>除去上清液，并在500微升磷酸盐缓冲盐水的悬浮珠粒（PBS）中-bovine血清白蛋白（BSA）-polyoxyethylenesorbitan单月桂酸酯（吐温20）钠的叠氮化物（PBS-TBN）通过涡旋和超声处理pH为7.4。沉淀通过微量离心珠以10,000 xg离心2分钟，去除上清。 注意:叠氮化钠是一种剧毒化学品。吞咽或与皮肤接触得到它是致命的。不要吸入粉尘/烟/气体/烟雾/蒸气或喷雾。穿戴合适的个人防护装备（PPE的）使用时，并按照相应的法律处置。 </li><li>悬浮通过涡旋和超声处理在1ml的PBS-TBN的珠子，持续20秒。 </li><li>沉淀通过微量离心珠以10,000 xg离心2分钟。 </li><li>重复步骤2.5和2.6。 注:这提供了一个共洗两次用PBS-TBN的。 </li><li>悬浮耦合和洗涤珠在1ml的PBS，1％BSA，0.05％叠氮化物，pH为7.4。存储偶联珠在黑暗中2-8°C冰箱。 </li></ol> 3.珠计数<ol><li>制备1:10稀释在水中或PBS缓冲液的偶联珠。 </li><li>负载10微升珠稀释到在样品引入点血球。 </li><li>计数在4×4角网格之一看到的珠子。计算使用下面的公式偶联珠的总数:计数（4×4栅格的1角）×（1×10 4）×（稀释因子）中毫升×悬浮体积。 </li></ol> 4.确认抗原耦合<ol><li>悬浮的通过涡旋和超声处理耦合到感兴趣的抗原，持续20秒珠的库存混合物。 </li><li>通过稀释偶联珠股最后准备工作珠的混合物100珠/μl的每个唯一珠在PBS-1％BSA的缓冲液（PBS-1％BSA，pH 7.4）中设定的浓度。 </li><li>准备根据制造商的在用PBS-1％BSA缓冲液96孔圆底板建议至少7两倍抗物种的系列稀释的IgG初级抗体。 </li><li>预湿的96孔过滤器底部板用100μl洗涤缓冲液的各抗原耦合确认试验的一个独立的8孔柱（8行），并通过真空除去上清液。加入50μl珠混合物（抗原偶联珠）合作，以预湿孔中。 </li><li>加入50μl抗体稀释到96孔过滤板的各列的行1-7和50微升的PBS-1％BSA缓冲液的排8 代替稀释抗体与作为背景孔。通过上下吹打5次用多通道移液器混合。每抗原偶联珠集执行相同的程序予以确认。 </li><li>盖，并允许在室温下在黑暗中孵育erature上在500rpm微孔板摇床上1小时。通过真空去除上清液。 </li><li>用100微升洗涤液洗井和真空上清。重复1X。重悬在50μl的PBS-1％BSA中的珠用多通道移液器。 </li><li>稀释生物素化抗物种特异性IgG二次检测抗体以在PBS-1％BSA的16微克/毫升。 </li><li>加入50μl稀释的二级抗体，以每孔移液器上下5次。 </li><li>覆盖过滤板，并允许在室温下在黑暗中孵育板振荡器上30分钟。通过真空去除上清液。用100微升洗涤液洗井和真空上清。重复洗1X。 </li><li>重悬在50μl的PBS-1％BSA中的珠用多通道移液器。 </li><li>稀释链霉亲和R-藻红蛋白记者（SAPE），以在PBS-1％BSA的24​​微克/毫升。 </li><li>加入50微升记者，每孔和上下吹打5次混匀。 </li><li> C过过滤板，并允许在室温下在黑暗中孵育板振荡器上30分钟。通过真空去除上清液。用100微升洗涤液洗井和真空上清。重复洗1X。 </li><li>在100μlPBS-1％BSA中的悬浮珠粒和分析使用分析仪29 50微升。 注意:基于珠子的多重免疫测定的结果中的中位荧光强度（MFI）进行测定。请务必参阅软件手册的最新版本，如果可避免的错误。 </li></ol> 5.唾液多重免疫测定<ol><li>从-80℃冰箱中取出唾液，并允许在室温下解冻。 </li><li>悬浮抗原通过涡旋和超声处理20秒偶联珠的股票。 </li><li>通过稀释偶联珠股100珠/μl的每个唯一珠集的在PBS-1％BSA的缓冲液中的终浓度制备工作珠混合物。 </li><li>准备1:唾液稀释4W¯¯第i个在96 PBS-1％BSA缓冲好，深孔板。 </li><li>预湿过滤板用100μl洗涤缓冲液中，用真空除去上清液。 </li><li>加入50μl工作珠混合物和稀释唾液等体积的95孔中对1的96孔过滤板的:8最终稀释。混合用多通道移液器反应。到了一个控制均匀，加入50微升抗原偶联珠加PBS-1％BSA缓冲液的50微升（如唾液替代）。 </li><li>盖，并允许在500rpm在室温下在黑暗中孵育1小时对微孔板摇床上。通过真空去除上清液。用100微升洗涤液洗井和真空上清。重复洗1X。 </li><li>悬浮珠粒于50μl的PBS-1％BSA中的与多通道移液器。 </li><li>稀释生物素化的山羊抗人IgG第二检测抗体在PBS-1％BSA的16微克/毫升。 </li><li>加入50μl稀释的第二抗体，每孔内容与混合多通道移液器。 </li><li>覆盖过滤板，并允许在室温下在黑暗中孵育板振荡器上30分钟。通过真空去除上清液。用100微升洗涤液洗井和真空上清。重复洗1X。 </li><li>悬浮珠粒于50μl的PBS-1％BSA中的与多通道移液器。 </li><li>稀释链霉亲和R-藻红蛋白记者（SAPE），以在PBS-1％BSA的24​​微克/毫升。 </li><li>加入50μl记者到各孔，并用多通道移液器混合。 </li><li>覆盖过滤板，并允许在室温下在黑暗中孵育板振荡器上30分钟。通过真空去除上清液。用100微升洗涤液洗井和真空上清。重复洗1X。 </li><li>在100μlPBS-1％BSA中的悬浮珠粒和分析使用分析仪29 50微升。 注:多重免疫测定的结果中的中位荧光强度（MFI）为单位进行测量。请务必参阅软件手册的最新版本，如果有的话，以避免错误。 </li></ol>

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US EPA Report Number: EPA/600/R-10/168: US EPA2011. , (2011).</li><li>Fujii, Y., 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=Serological+surveillance+development+for+tropical+infectious+diseases+using+simultaneous+microsphere-based+multiplex+assays+and+finite+mixture+models.">Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.</a> PLoS Negl Trop Dis. 8, e3040 (2014).</li><li>Rota, M., Massari, M., Gabutti, G., Guido, M., De Donno, A., Ciofi degli Atti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measles+serological+survey+in+the+Italian+population:+interpretation+of+results+using+mixture+model.">Measles serological survey in the Italian population: interpretation of results using mixture model.</a> Vaccine. 26 (34), 4403-4409 (2008).</li><li>Vyse, A., Gay, N., Hesketh, L., Pebody, R., Morgan-Capner, P., Miller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+serological+surveys+using+mixture+models:+the+seroepidemiology+of+measles,+mumps+and+rubella+in+England+and+Wales+at+the+beginning+of+the+21st+century.">Interpreting serological surveys using mixture models: the seroepidemiology of measles, mumps and rubella in England and Wales at the beginning of the 21st century.</a> Epidemiol Infect. 134 (6), 1303-1312 (2006).</li><li>Baughman, A., 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=Establishment+of+diagnostic+cutoff+points+for+levels+of+serum+antibodies+to+pertussis+toxin,+filamentous+hemagglutinin,+and+fimbriae+in+adolescents+and+adults+in+the+United+States.">Establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the United States.</a> Clin Diagn Lab Immunol. 11 (6), 1045-1053 (2004).</li><li>Clotilde, L., Bernard, C., Hartman, G., Lau, D., Carter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbead-based+immunoassay+for+simultaneous+detection+of+Shiga+toxins+and+isolation+of+Escherichia+coli+O157+in+foods.">Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.</a> J Food Prot. 74 (3), 373-379 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Luminex User Software Manual (RUO) xPONENT 3.1 Rev. 2. 3, 6-102 (2014).</li><li>Waterboer, T., Sehr, P., Pawlita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+non-specific+binding+in+serological+Luminex+assays.">Suppression of non-specific binding in serological Luminex assays.</a> J Immunol Methods. 309 (1-2), 200-204 (2006).</li></ol>

美国环境保护署通过其研究和发展办公室资助和管理这里所描述的研究。它一直受到机构的行政审查和批准发布。商品名称或商品的提及，并不构成认可或推荐使用。

这些结果表明，在多重免疫测定方法是用于唾液样品是免疫阳性或免疫阴性区分是有用的。为了确定免疫阳性，单个切点通过计算平均值加上的与所有的唾液样品的测试的控制耦合珠粒的日志变换MFI响应的三个标准偏差的发展。截止点，得到以评估曝光和immunoprevalence到单个或多种病原体的能力。这种辨别力可能是调查与接触环境和其他病原体相关的健康风险在人口研究非常有用。 <p class="jove_c...

腹泻相关的疾病的百分之八十八世界范围内与人体接触到受污染的水，不安全的食品和卫生条件差/卫生有关，造成约1.5万人死亡，其中大多数是儿童1。这是公共卫生官员和决策者关注的一个重要原因。在努力，调查与水性和其他环境中的病原体相关风险和疾病，我们开发了多重免疫测定来衡量人类样本2-4的抗体。这种方法可以适用于流行病学研究，以确定人体暴露于这些病原体和更好地确定immunoprevalence和事件感染。 唾液持有相当大的希望作为替代血清人类生物标志物研究。间使用唾液的优点是，非侵入性和易于样品收集，成本低的，并且样品可以容易地从儿童5-7 &lt;收集/ SUP&gt;。血清和唾液样本已经被反对H.抗体广泛的研究幽门螺杆菌 2,3,8， 恶性疟原虫 9，阿米巴 10，隐孢子虫 3,11， 肺炎链球菌 12，肝炎病毒A和C 13-14， 诺如病毒 2-4,15，T。弓形虫 2-4，登革热病毒16，人免疫缺陷病毒（HIV）17，和大肠杆菌 O157:H7 18。 一个多重免疫测定允许在单个样本量同时和一个周期或运行中的多个分析物的分析。从C抗原复空肠弯曲菌 ，T。弓形虫，幽门螺旋杆菌，A型肝炎病毒，和两个诺如病毒被用来测量人体唾液的IgG 2-4和IgA 3,4-和血浆的IgG 2,3抗体反应使用这些病原体珠为基础的复用免疫。当在暴露于微生物的水，土壤和食品的流行病学研究结合使用，在此研究中所描述测定的类型可以提供有价值的信息，以提高所造成的环境中的病原体感染的理解。而且，从这些研究获得的唾液抗体的数据可以被用来改善风险评估模型19-22。

<table><tbody><tr><td>&lt;strong&gt;Equipment and Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Electron Corporation</td><td>75002446</td><td>Used to centrifuge samples</td></tr><tr><td>Vortex Mixer</td><td>VWR</td><td>G560</td><td>Used to mix samples</td></tr><tr><td>Sonicator (mini)</td><td>Fisher Scientific</td><td>15-337-22</td><td>Used to separate beads</td></tr><tr><td>Pipettors P10, P20, P100, P1000, 8 ch.</td><td>Capp</td><td>Various</td><td></td></tr><tr><td>Hemacytometer (Bright Line)</td><td>Housser Scientific&amp;nbsp;</td><td>3200</td><td>Used to count coupled beads</td></tr><tr><td>Multiscreen Vacuum Manifold</td><td>Millipore</td><td>MSVMHTS00</td><td>Used in washing steps to remove supernatant</td></tr><tr><td>MicroShaker</td><td>VWR</td><td>12620-926</td><td>Used to agitate beads during incubations</td></tr><tr><td>Tube rack (1.5 ml and 0.5 ml) (assorted)</td><td>VWR</td><td>30128-346</td><td></td></tr><tr><td>Weighing Scale</td><td>Mettler or other</td><td></td><td>Used to measure wash reagents for making buffers</td></tr><tr><td>Dynabead Sample Mixer</td><td>Invitrogen</td><td>947-01</td><td>Used during coupling incubation step</td></tr><tr><td>MatLab (R2014b)</td><td>The MathWorks, Inc.</td><td></td><td>Used to analyze antibody response data</td></tr><tr><td>Microsoft Excel 2014</td><td>Microsoft Corporation</td><td></td><td>Used to analyze antibody response data</td></tr><tr><td>Luminex Analyzer with xPonent 3.1 software</td><td>Luminex Corporation</td><td>LX200-XPON3.1</td><td>Instrument and software used to run assay</td></tr><tr><td>&lt;strong&gt;Antigens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GI.1 Norwalk Virus: p-particle</td><td>Xi Jiang (CCHMC)*</td><td>NA</td><td>*Cincinnati Childrens&#039; Hospital. Final conc. 5 &amp;micro;g.</td></tr><tr><td>GII.4 Norovirus VA387: p-particle</td><td>Xi Jiang (CCHMC)*</td><td>NA</td><td>*Cincinnati Childrens&#039; Hospital. Final conc. 5 &amp;micro;g.</td></tr><tr><td>Hepatitis A Virus: grade II concentrate from cell culture</td><td>Meridian Life Sciences</td><td>8505</td><td>Antigen coupled at 100 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Helicobacter pylori&lt;/em&gt;: lysate</td><td>Meridian Life Sciences</td><td>R14101</td><td>Antigen coupled at 25 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Toxoplasma gondii&lt;/em&gt;: recombinant p30 (SAG1)</td><td>Meridian Life Sciences</td><td>R18426</td><td>Antigen coupled at 25 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Campylobacter jejuni&lt;/em&gt;: heat killed whole cells</td><td>KPL</td><td>50-92-93</td><td>Antigen coupled at 50 &amp;micro;g</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Guinea pig anti-Norovirus</td><td>(CCHMC)*</td><td>NA</td><td>Used for coupling confirmation</td></tr><tr><td>Mouse anti-Hepatitis A IgG</td><td>Meridian Life Sciences</td><td>C65885M</td><td>Used for coupling confirmation</td></tr><tr><td>Mouse anti-Hepatitis A IgG</td><td>Meridian Life Sciences</td><td>C65885M</td><td>Used for coupling confirmation</td></tr><tr><td>BacTraceAffinity Purified Antibody to &lt;em&gt;Helicobacter pylori&lt;/em&gt;</td><td>KPL</td><td>01-93-94</td><td>Used for coupling confirmation</td></tr><tr><td>Goat pAb to&lt;em&gt; Toxoplasma gondii&lt;/em&gt;</td><td>Abcam</td><td>Ab23507</td><td>Used for coupling confirmation</td></tr><tr><td>BacTrace Goat anti-&lt;em&gt;Campylobacter&lt;/em&gt; species</td><td>KPL</td><td>01-92-93</td><td>Used for coupling confirmation</td></tr><tr><td>&lt;strong&gt;Secondary&amp;nbsp; Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L)</td><td>Jackson</td><td>705-065-149</td><td>Used for coupling confirmation</td></tr><tr><td>Biotinylated Rabbit anti-Goat IgG (H+L)</td><td>KPL</td><td>16-13-06</td><td>Used for coupling confirmation</td></tr><tr><td>Biotinylated Goat anti-Mouse IgG (H+L)</td><td>KPL</td><td>16-18-06</td><td>Used for coupling confirmation</td></tr><tr><td>Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG (H+L)</td><td>KPL</td><td>176-1506</td><td>Used for coupling confirmation</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 ml copolymer microcentrifuge tubes</td><td>USA Scientific</td><td>1415-2500</td><td>Used as low binding microcentrifuge tubes</td></tr><tr><td>10 &amp;micro;l pipette tip refills</td><td>BioVentures</td><td>5030050C</td><td></td></tr><tr><td>200 &amp;micro;l pipette tip refills</td><td>BioVentures</td><td>5030080C</td><td></td></tr><tr><td>1,000 &amp;micro;l pipette tip refills</td><td>BioVentures</td><td>5130140C</td><td></td></tr><tr><td>Aluminum foil</td><td>Various Vendors</td><td></td><td>Used keep beads in the dark during incubations</td></tr><tr><td>Deep Well plates</td><td>VWR</td><td>40002-009</td><td>Used for diluting saliva samples</td></tr><tr><td>Multiscreen Filter Plates</td><td>Millipore</td><td>MABVN1250</td><td>Used to run assays</td></tr><tr><td>Oracol saliva collection system</td><td>Malvern Medical Developments Limited</td><td></td><td>Used for saliva collection</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carboxylated microspheres (beads)</td><td>Luminex Corporation</td><td>Dependent on bead set</td><td>Antigens are coupled to the microspheres</td></tr><tr><td>EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)</td><td>Pierce</td><td>77149 or 22980</td><td>Used in bead activation</td></tr><tr><td>Sulfo-NHS (&lt;em&gt;N&lt;/em&gt;-hydroxysulfosuccinimide)</td><td>Pierce</td><td>24510</td><td>Used in bead activation</td></tr><tr><td>Steptavidin-R-phycoerythrin (1 mg/ml)</td><td>Molecular Probes</td><td>S-866</td><td>Used as reporter</td></tr><tr><td>MES (2-[&lt;em&gt;N&lt;/em&gt;-Morpholino]ethanesulfonic acid)</td><td>Sigma</td><td>M-2933</td><td>Used for coupling</td></tr><tr><td>Tween-20 (Polyoxyethylenesorbitan monolaurate)</td><td>Sigma</td><td>P-9416</td><td>Used in wash buffer to remove non-specific binding</td></tr><tr><td>&lt;strong&gt;Protein Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)&lt;/strong&gt;**</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;C</td></tr><tr><td>PBS, pH 7.4</td><td>Sigma</td><td>P-3813</td><td>138 mM NaCl, 2.7 mM KCl</td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7888</td><td>0.1% (w/v)</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P-9416</td><td>0.2% (v/v)</td></tr><tr><td>Sodium Azide (0.05% azide)**</td><td>Sigma</td><td>S-8032</td><td>**&lt;strong&gt;Caution&lt;/strong&gt;: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE&#039;s. Dispose of according to applicable laws.</td></tr><tr><td>&lt;strong&gt;MES/Coupling Buffer (0.05 M MES, pH 5.0)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MES (2-[&lt;em&gt;N&lt;/em&gt;-Morpholino]ethanesulfonic acid)</td><td>Sigma</td><td>S-3139</td><td></td></tr><tr><td>5 N NaOH</td><td>Fisher</td><td>SS256-500</td><td></td></tr><tr><td>&lt;strong&gt;Assay Buffer (PBS, 1% BSA, pH 7.4)&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;C</td></tr><tr><td>PBS, 1% BSA, pH 7.4</td><td>Sigma</td><td>P-3688</td><td>138 mM NaCl, 2.7 mM KCl, 1% BSA</td></tr><tr><td>&lt;strong&gt;Activation Buffer (0.1 M NaH&lt;/strong&gt;&lt;sub&gt;&lt;strong&gt;2&lt;/strong&gt;&lt;/sub&gt;&lt;strong&gt;PO&lt;/strong&gt;&lt;sub&gt;&lt;strong&gt;4&lt;/strong&gt;&lt;/sub&gt;&lt;strong&gt;, pH 6.2)&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;C</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Sodium phosphate, monobasic anhydrous)</td><td>Sigma</td><td>S-3139</td><td>0.1 M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>5 N NaOH</td><td>Fisher</td><td>SS256-500</td><td></td></tr><tr><td>&lt;strong&gt;Wash Buffer (PBS, 0.05% Tween-20, pH 7.4)&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;C</td></tr><tr><td>PBS, 0.05% Tween-20, pH 7.4</td><td>Sigma</td><td>P-3563</td><td>138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN</td></tr></tbody></table>

developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens

其病因与人体暴露于环境中的病原体的影响是全球主要的关注，因此，评估接触和感染的使用成本效益，高通量方法的能力是不可缺少的。该原稿描述了能够同时测量抗体在人唾液的存在下，以多种病原体的基于珠子的多重免疫测定的开发和分析。唾液是在本申请中特别有吸引力的，因为它是无创，更便宜和更容易收集比血清。从环境中的病原体的抗原偶联到羧基化微球（珠）和用于测量使用基于珠子的，溶液相测定法在人唾液样品的非常小体积的抗体。珠再加上从空肠弯曲菌，幽门螺旋杆菌，弓形虫，诺如病毒（G I.1和G II.4）抗原，甲型肝炎病毒。以保证抗原充分耦合到珠，耦合用物种特异性的，动物来源的初级捕获抗体证实，随后用生物素化抗物种二次检测抗体和链霉抗R-藻红蛋白记者（SAPE）温育。作为衡量的非特异性结合的对照，一个小珠组被同样处理的其他不同的是它不连接到任何抗原。抗原耦合和控制珠子然后用前瞻性收集的人唾液样品孵育，基于流式细胞术的原理的高通量分析仪测量，和抗体的每个抗原的存在下，在中位数荧光强度单位测定（MFI） 。这个多重免疫测定具有许多优点，包括具有较少样本更多的数据;降低了成本和劳动;并能够自定义检测到许多感兴趣的目标。结果表明，该唾液多重免疫测定可以是能够识别先前接触和感染的，它可以是especiaLLY有用的涉及大型人群监测研究。

一个唯一的珠集被用来作为测量非特异性结合对照和样品采样变性。这些珠粒相同处理，以与异常的抗原偶联珠，他们不与在偶合步骤中的任何抗原温育。从所有的唾液样本培养控制珠获得MFI值&gt; 500被拆除，由于从进一步分析，从血清中怀疑被污染，其余的答复日志分配。唾液可以与血清污染如果牙龈与附连到收集装置，或者如果在研究参与者都有牙周病海绵擦太剧烈?...

Watch this Scientific Journal Video about Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens at JoVE.com

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

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 ...

developing-salivary-antibody-multiplex-immunoassay-to-measure-human

Research

JoVE Journal

Immunology and Infection

8.7K 次观看.  U.S. Environmental Protection Agency. 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.

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

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

科研

化学

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

生物工程

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

免疫与感染

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

基于磁珠复合法的撕裂细胞因子分析

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

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 animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

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 ...

生物学

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

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 ...

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

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复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

无交叉反应和无免疫多路复用夹芯芯片

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

基于磁珠复合法的撕裂细胞因子分析

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

复用荧光免疫检测方法和故障排除

使用流感抗原微阵列测量病毒亚型血清抗体的广度

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

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