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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment and Software</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&#160;</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 (1.5mL and 0.5mL) (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>Antigens</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>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;g.</td>
		</tr>
		<tr>
			<td>GII.4 Norovirus VA387 : p-particle</td>
			<td>Xi Jiang (CCHMC)*</td>
			<td>NA</td>
			<td>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;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 &#181;g</td>
		</tr>
		<tr>
			<td>Helicobacter pylori : lysate</td>
			<td>Meridian Life Sciences</td>
			<td>R14101</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Toxoplasma gondii : recombinant p30 (SAG1)</td>
			<td>Meridian Life Sciences</td>
			<td>R18426</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Campylobacter jejuni : heat killed whole cells</td>
			<td>KPL</td>
			<td>50-92-93</td>
			<td>Antigen coupled at 50 &#181;g</td>
		</tr>
		<tr>
			<td>Primary Antibodies</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 Helicobacter pylori</td>
			<td>KPL</td>
			<td>01-93-94</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Goat pAb to Toxoplasma gondii</td>
			<td>Abcam</td>
			<td>Ab23507</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>BacTrace Goat anti-Campylobacter species</td>
			<td>KPL</td>
			<td>01-92-93</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Secondary&#160; Antibodies</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>Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(&#7518;)&#160;</td>
			<td>KPL</td>
			<td>16-10-02</td>
			<td>Used for Salivary Immunoassay</td>
		</tr>
		<tr>
			<td>Consumables</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 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030050C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030080C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;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>Reagents</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-[3dimethylaminopropyl] carbodiimide hydrochloride)</td>
			<td>Pierce</td>
			<td>77149 or 22980</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Pierce</td>
			<td>24510</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Steptavidin-R-phycoerythrin (1mg/mL)</td>
			<td>Molecular Probes</td>
			<td>S-866</td>
			<td>Used as reporter</td>
		</tr>
		<tr>
			<td>MES (2-[N-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>Protein Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)**</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;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>**Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE&#39;s. Dispose of according to applicable laws.</td>
		</tr>
		<tr>
			<td>MES/ Coupling Buffer (0.05 M MES, pH 5.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MES (2-[N-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>Assay Buffer (PBS, 1% BSA, pH 7.4)&#160;</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;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>Activation Buffer (0.1 M NaH2PO4, pH 6.2)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaH2PO4 (Sodium phosphate, monobasic anhydrous)</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td>0.1M NaH2PO4</td>
		</tr>
		<tr>
			<td>5 N NaOH</td>
			<td>Fisher</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer (PBS, 0.05% Tween-20, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

一种测试洗手方法去除新发感染性病原体的方法

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

细胞 Bead-based 免疫分析平台对受激小鼠脾多重细胞因子的研究

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...