作者想感谢他们的合作和后勤支持，罗马大学“的Tor Vergata”分析化学实验室南希唐纳和所有成员。这项工作是支持由欧盟项目“BioCop”。

1）阻塞包被的磁珠： 继续如下块包被的磁珠： <ol><li>准备一个2毫升Eppendorf管; </li><li>均质（摇晃，但没有涡旋）包被的磁珠（见附录准备包被的磁珠）使用样品旋转的搅拌机; </li><li>紧随之后，同质化，移液器10μL到每个单独的Eppendorf管包被的磁珠; </li><li>添加脱脂阻止到Eppendorf管中设置的每个解决方案的牛奶1毫升; </li><li>让我们为在室温下30分钟孵育旋转样品混合器磁珠; </li><li>取出阻塞的解决方案：这样做2分钟（见管侧壁上坚持磁珠）的磁性部分磁粉浓缩管;仔细移液器关闭上清，留下珠不受干扰; </li><li>加入1 ml PBS缓冲液+ NaN的3 + BSA作为存储解决方案，为每个管中; </li><li>商店涂层和封锁的磁珠在4 ° C（请注意，包被的磁珠至少2个月稳定在4 ° C）; </li></ol> 2）免疫磁珠链建设的校准曲线和样品分析继续为有准备的测量磁珠： <ol><li>以涂层和阻止你要分析每个样品中提取的解决方案之一的Eppendorf管（2毫升）; </li><li>以一个额外的Eppendorf管中，每一个工作的校准解决方案; </li><li>均质磁珠，使用前的旋转样品混合器2分钟; </li><li>删除存储磁粉浓缩液使用; </li><li>与PBS / BSA洗涤液洗磁珠三次。删除洗涤液; </li><li> 比赛步 ：添加每个包含管洗涤磁珠，200μL样品提取准备;每个样品使用一个磁珠管。每个标准溶液，重复同样的程序。单克隆抗体溶液中加入200μL，每个磁珠管。让磁珠孵育半小时旋转样品在室温下搅拌机; </li><li> Eppendorf管中取出的竞争步使用的解决方案; </li><li> 贴标步：每个磁珠Eppendorf管中添加标记二抗400μl。让磁珠孵育半小时旋转样品在室温下搅拌机; </li><li> Eppendorf管中删除标签解决方案; </li><li> 3次，PBS /吐温® 20洗涤液洗磁珠。删除的液体; </li><li>悬浮与DEA的缓冲区100μL每个等份磁珠。现在磁珠准备电化学测量步骤; </li></ol> 3）大会PalmSens多路复用器（MUX）选项请注意：底物水解酶产品犯规电极表面，这样做的原因为每个测量都需要一个新的电极。 <ol><li>将PC连接笔记本电脑通过串行电缆PalmSens; </li><li> CH8与PalmSens复用器连接9 - DIN插头。 </li><li>八通道MUX CH8的多路复用器的电接触连接的串行电缆; </li><li>广场上的8个磁铁块电极地带。注意每个磁铁放置在每一个工作电极（注意，需要为每个电极的单磁铁是势在必行，否则磁粉不会在工作电极面积集中）; </li><li>插入电极地带到8通道MUX电接点; </li></ol>在相应的框中使用DPV测量以下参数： 个人开始：0（V），E端：0.6（V）; E步骤：0.016（V）; E脉冲：0.0339（V），E调理：0（V），E沉积：0（V），扫描速度：0.1（V / S），T脉冲：0.06（S），T空调：0（S），T沉积：0（S），T平衡：8（S）。 4）酶反应和电化学测量<ol><li>均质通过轻轻晃动每个Eppendorf管中包含为了准备电化学测量步骤磁珠，获得了分散良好的悬浮磁珠; </li><li>移液器从每Eppendorf公司后，立即同质化，分装20μL到相应的工作电极表面的磁性珠分散。对于每一个地带使用的电极20μL磁珠取自不同的Eppendorf管（例如8个电极使用八个不同的Eppendorf）; </li><li>第一电极上加入80μL的酶底物溶液。开始2分钟倒计时。 14秒后，加入80μl酶底物溶液的第二电极。另外14秒后，添加第三个电极上的酶底物溶液80μL。继续在同一时间INTerval（14秒），直到最后电极（这意味着：掉落每间隔14秒的基板等分）。 14秒的间隔时间的计算公式为电位进行测量所需的时间;注意，80μL每个传感器的解决方案应包括工作，参考电极。此外，每个传感器的解决方案不应接触到相邻电极的解决方案。 </li><li> 2分钟后计数的酶底物溶液此外，启动电化学测量; </li><li>使用PalmSens Lite软件获得的峰值电流（μA），为每个解决方案。 </li></ol> 5）计算使用后勤4个参数方程建立校准曲线，并计算在总HT-2/T-2毒素量浓度微克每毫升含有未知样品每个Eppendorf公司管。 实验数据，对校准物的浓度（ng / ml的）的峰高（μA），必须安装使用一种非线性的后勤（4 - PL）四参数方程图（1）（使用适马剧情8.0或Kaleidagraph）。通常采用4 - PL的非线性模型来形容的配体结合试验（的LBA）6 。 <img src="/files/ftp_upload/1588/1588eq1.jpg" alt="公式1" /> （1） A，B，X0，Y0后勤参数Sigma的绘制曲线拟合程序。 使用阐释的公式来计算的稀释后的样品提取液HT-2/T-2毒素总量的内容 <img src="/files/ftp_upload/1588/1588eq2.jpg" alt="公式（2）" /> （2） x是在稀释后的提取液，后勤4个参数方程每milliltre纳克（ng / ml的）计算，总金额HT-2/T-2毒素的质量浓度 y是μA电流值未知样品获得附录 6）包被的磁珠继续作为包被的磁珠： 洗涤程序 <ol><li>均质tosylactivated磁珠® M - 280（2 × 109珠/毫升原液）摇晃和震荡（最大速度为1分钟（避免起泡）（请注意磁珠的浓度需求将始终是相同的，以确保可重复性在）测量）; </li><li>立即到2 mL Eppendorf管吸管1毫升以上匀浆珠; </li><li> 2分钟（见管侧壁上坚持磁珠）的磁性部分放置在磁粉浓缩管; </li><li>仔细移液器关闭上清，留下珠不受干扰; </li><li>从磁粉集中删除Eppendorf管中，重悬在1毫升0.1M的硼酸盐缓冲液pH值9.5的珠子。轻轻混匀，在2分钟旋转样品混合器2分钟; </li><li>移液器关闭上清; </li><li>吸取1毫升的硼酸缓冲液的Eppendorf管。磁珠现在准备涂层步骤; </li></ol> 涂层过程 <ol start="8"><li> KLH（匙孔戚血蓝蛋白）原液1毫升磁珠）（HT - 2 - KLH最终浓度为375毫克/毫升）加入600毫升的HT - 2共轭;孵化磁珠和HT - 2 - KLH解决方案20H在37 ° C *缓慢倾斜旋转旋转样品混合器; </li><li>经过孵化的地方磁粉浓缩和移液器关闭上清管; 用1毫升PBS / BSA缓冲液洗涂珠两次。彼此之间洗涤除去PBS / BSA溶液。设置为5分钟的旋转搅拌机磁珠和PBS / BSA的所有洗涤步骤进行; </li><li>使用1毫升的TRIS / BSA缓冲液洗一次磁珠。删除的TRIS / BSA溶液。开展这一步，通过磁珠的TRIS / BSA缓冲设置为4小时旋转搅拌机在37 ° C *; </li><li>磁珠洗一次使用1毫升刷新（在冰箱保存在4 ° C）的PBS / BSA缓冲液。删除PBS / BSA溶液。开展这一步骤设置磁珠和PBS / BSA缓冲液在室温5分钟旋转的搅拌机; </li><li>经过洗涤步骤删除所有的洗涤液，并添加1 ml PBS缓冲液中+为NaN 3 + BSA作为贮存液体; </li><li>商店包被的磁珠在4 ° C（请注意，包被的磁珠至少2个月稳定在4 ° C）; </li></ol> *为了这个目的，混频器是放置在烤箱配备了一个洞，允许插入电源电缆。或者混频器可放置于37℃在恒温室 7）样品制备和提取 25克磨细的样品（婴儿食品或早餐谷物），我们ighed成一个搅拌杯，用100毫升的乙腈水溶液（一十四分之八十六）/ 3分钟在高速搅拌器提取。在离心5分钟4000转（3000克），8毫升上清和Mycosep柱纯化后，4毫升清洗提取干下氮气流。干燥样品，可以储存在-30 ° C到几个月不等。 8）样品重组 早餐谷物 重组干早餐谷物提取物（谷物为基础的食品，对成人消费注定）与40毫升的PBS pH值7.4。这样等于所谓的法律限制（200纳克/克）的毒素浓度的样本将属于工作范围的中间一个信号，测量步骤。 婴儿食品 重组干的婴儿食品提取物（谷物为基础的食品，对婴幼儿消费注定）4毫升的PBS。这样等于所谓的法律限制（20-25毫微克/克）的毒素浓度的样本将属于工作范围的中间一个信号，测量步骤。

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	<li>Koch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+of+trichothecenes+analysis.">State of the art of trichothecenes analysis.</a> Toxicol. Letters. 153, 109-112 (2004).</li><li>Krska, R., Baumgartner, S., Josephs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+in+the+analysis+of+type-A+and-B+trichothecene+mycotoxins+in+cereals.">State of the art in the analysis of type-A and-B trichothecene mycotoxins in cereals.</a> Fresenius J. Anal. Chem. 371, 285-299 (2001).</li><li>Morozova, T. Y., Morozov, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+differentiation+in+recognition+of+cross-reactive+antigens+by+magnetic+beads.">Force differentiation in recognition of cross-reactive antigens by magnetic beads.</a> Anal. Biochem. , 263-271 (2008).</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=FAO+Food+and+Nutrition+Paper+74.">FAO Food and Nutrition Paper 74.</a> JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES Fifty-sixth meeting. , 115 (2001).</li><li>Piermarini, S., Volpe, G., Ricci, F., Micheli, L., Moscone, D., Palleschi, G., Fuhrer, M., Krska, R., Baumgartner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Screening+Electrochemical+Methods+for+Aflatoxin+B1+and+Type-A+Trichothecenes:+a+preliminary+study.">Rapid Screening Electrochemical Methods for Aflatoxin B1 and Type-A Trichothecenes: a preliminary study.</a> Anal. Lett. 40, 1333-1346 (2007).</li><li>Findlay, J. W. A., Dillard, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriate+calibration+curve+fitting+in+ligand+binding+assays.">Appropriate calibration curve fitting in ligand binding assays.</a> AAPS J. 9 (2), E260-E267 (2007).</li><li>Ricci, F., Volpe, G., Micheli, l., Palleschi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+novel+developments+and+applications+of+immunosensors+in+food+analysis.">A review on novel developments and applications of immunosensors in food analysis.</a> Anal. Chim. Acta. 605 (2), 111-129 (2007).</li></ol>

利用抗体作为生物分子识别探头已经出现了遥感技术的广泛使用;免疫组织化学检测方法，如酶联免疫吸附和MEIAs，时下许多实验室 7中最常用和应用平台之间， 。 虽然这些方法在过去几年中实现特殊的敏感性和特异性，许多研究小组的主要目标，已被其表演的改进和优化。 在这个协议中，我们已经展示了如何编造并询问霉菌毒素的检测的电化学?...

试剂<table border="1" cellspacing="0" cellpadding="0"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P9333 </td><td></td></tr><tr><td>磷酸二氢钾</td><td>西格玛</td><td> P9791 </td><td></td></tr><tr><td>磷酸氢二钠</td><td>西格玛</td><td> S3264 </td><td></td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S3014 </td><td></td></tr><tr><td> MgCl 2的无水</td><td>西格玛</td><td> M8266 </td><td></td></tr><tr><td> DEA（99.5％） </td><td>西格玛</td><td> 31589 </td><td></td></tr><tr><td> HCL 37％ </td><td>西格玛</td><td> 320331 </td><td></td></tr><tr><td> H 3 BO 3 </td><td>西格玛</td><td> B6768 </td><td></td></tr><tr><td>三羟甲基]氨基甲烷</td><td>西格玛</td><td> 252859 </td><td></td></tr><tr><td> BSA（牛血清白蛋白） </td><td>西格玛</td><td> A4503 </td><td></td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> S5881 </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P9416 </td><td></td></tr><tr><td>为NaN 3 </td><td>爱秩序</td><td> 71290 </td><td></td></tr><tr><td> 1 - 萘基磷酸二钠</td><td> Fluka公司</td><td> N7255 </td><td></td></tr><tr><td>脱脂牛奶封闭液 - 非脱脂奶粉</td><td> Bio - Rad公司</td><td> 170-6404 </td><td></td></tr><tr><td> HT - 2共轭与KLH（匙孔戚血蓝蛋白），储备溶液（1毫克/毫升的PBS）： </td><td> Biopure </td><td> 004050 </td><td> HT - 2 - KLH偶联物，得到了由N，N&#39; -羰基（​​CDI）和激活的HT - 2毒素，激活位置3和4的HT - 2毒素的OH - CDI方法让反应蛋白（KLH）aminogroups产生一个稳定的carbammate联动。 </td></tr><tr><td>二次标记抗体：从马抗小鼠IgG（H + L），碱性磷酸酶标记，浓度为1毫克/毫升。 </td><td>媒介实验室</td><td> AP - 2000 </td><td></td></tr><tr><td> HT - 2毒素</td><td> Biopure </td><td></td><td></td></tr><tr><td>磁珠磁珠® </td><td> DYNAL </td><td> M - 280 Tosylactivated </td><td>浓度为2 × 10 9粒/毫升。 </td></tr></table>解决方案<table border="1" cellspacing="0" cellpadding="0"><tr><td> 试剂名称 </td><td> 制备 </td><td> 评论（可选） </td></tr><tr><td>磷酸盐缓冲液（PBS），pH值7.4 </td><td>氯化钾0.20克，0.20克磷酸二氢钾，磷酸氢二钠1.16克和8.00克氯化钠溶解在900毫升的水</td><td></td></tr><tr><td>二乙醇胺缓冲液，DEA，0.97中号+ 1毫米氯化镁2 + 0.15 M氯化钾，pH值9.8 </td><td> MgCl 2的无水，氯化钾和59年3月7日克〜300毫升的水溶解0.0476克。解散后，添加51毫升的DEA（99.5％）。调节pH值至9.8，用盐酸（6米）。与水稀释至500毫升。 </td><td></td></tr><tr><td>硼酸盐缓冲液，pH值9.5，0.1 M </td><td> H 3 BO 3〜300毫升的蒸馏水溶解3.09克;用NaOH和/或盐酸（6米或低浓度）的pH值调整至9.5。用蒸馏水稀释至500毫升。 </td><td></td></tr><tr><td> TRIS缓冲液，0.2 M，pH值8.5 </td><td>三羟甲基]在100毫升的水基氨基甲烷溶解3.85克。用NaOH和/或盐酸（6米或低浓度），调整pH至8.5。与水稀释至200毫升。 </td><td></td></tr><tr><td> TRIS缓冲液+ BSA溶液（0.1％），pH值8.4 </td><td>溶解在50毫升的Tris缓冲液pH值8.4的牛血清白蛋白的0.050克。 </td><td>这个解决方案必须使用当天新鲜配制。 </td></tr><tr><td> PBS缓冲液+吐温® 0.05％ </td><td>吐温20克溶解在500毫升先前准备的PBS缓冲液，pH值7.4 0.250 </td><td></td></tr><tr><td> PBS缓冲液+ BSA溶液0.1％ </td><td>溶解在50毫升的PBS缓冲液，pH值7.3的牛血清白蛋白的0.050克。 </td><td>这个解决方案必须使用当天新鲜配制。 </td></tr><tr><td> PBS缓冲液+ BSA 0.1％+南安3 0.02％ </td><td> BSA和NaN的3 0.010 0.050克溶解在50毫升的PBS缓冲液，pH值7.3。 </td><td>存储溶胶ution </td></tr><tr><td>酶法基板</td><td> 0.010克，1 - 萘磷酸钠盐溶解在100毫升的DEA缓冲液pH值9.8。铝箔紧紧包裹烧瓶。 </td><td>这个解决方案必须使用当天新鲜配制。 </td></tr><tr><td>脱脂牛奶封闭液</td><td>加入0.20克印迹级拦截器非脱脂奶粉（BIO - RAD，大力神，加利福尼亚，美国），以200毫升的PBS缓冲液pH值7.3。 </td><td>这个解决方案必须使用当天新鲜配制。 </td></tr><tr><td> HT - 2原液</td><td>溶解的单端孢HT - 2毒素小瓶10毫克，10毫升的乙腈给 与浓度为1毫克/毫升的解决方案。 </td><td>分割成30毫升的一次性使用的等分这一解决方案，并储存在低于-30 ° C </td></tr><tr><td> HT - 2工作标准溶液</td><td>移液器HT - 2毒素原液20毫升到2毫升校准容量瓶中，用乙腈稀释，以获得一个HT - 2的溶液中含有10微克/ ml的单端孢毒素。 </td><td>这个解决方案应该使用当天新鲜配制。 </td></tr><tr><td> HT - 2校准解决方案</td><td>稀释的HT - 2工作标准溶液（10微克/毫升）准备工作校准解决方案，100毫微克/毫升。稀释HT - 2的工作校准解决方案，100 ng / ml的准备工作以下浓度0（空白），0.5，1，2，4，10毫微克/毫升的校准解决方案。 </td><td>这些解决方案都必须准备使用当天新鲜。 </td></tr><tr><td> HT - 2共轭与KLH </td><td>分裂与KLH（匙孔戚血蓝蛋白）储备溶液（1毫克/毫升的PBS）HT - 2的共轭到350μL的一次性使用的等分。 </td><td>在-30 ° C店等分</td></tr><tr><td>特异性单克隆抗体</td><td>稀释1:350（V：V）的股票在PBS的浓度（1.383毫克/毫升）单克隆抗体的使用在竞争中一步</td><td>在使用的时候，这个解决方案必须准备新鲜。 10毫升的PBS抗体股票最终体积为3.5毫升的解决方案，足以分析17标准和/或样品。 </td></tr><tr><td>中学标记抗体</td><td>抗小鼠IgG（H + L）与碱性磷酸酶标记是1:100（V：V）的PBS稀释到使用在竞争中的一步。 </td><td>在PBS 100μL抗体股票最终体积为10毫升的解决方案，足以分析25标准和/或样品。在使用的时候，这个解决方案必须准备新鲜。 </td></tr></table>仪器<table border="1" cellspacing="0" cellpadding="0"><tr><td> 试剂名称 </td><td> 公司 </td><td> 评论（可选） </td></tr><tr><td>磁粉浓缩：MPC ® - S </td><td> DYNAL </td><td></td></tr><tr><td> PalmSens仪器</td><td> PalmSens </td><td>提供PalmSens建兴，串行电缆连接电脑笔记本电脑和软件复用器选项</td></tr><tr><td> CH8 PalmSens复用器</td><td> PalmSens </td><td></td></tr><tr><td>八通道MUX电接点</td><td>手工制作</td><td></td></tr><tr><td>地带与八个丝网印刷电极（SPES） </td><td>手工制作</td><td></td></tr><tr><td>特殊设计的电极地带的支持</td><td>手工制作</td><td>每个是放在下面的每一个工作电极表面的SPE包括8钕磁铁</td></tr><tr><td>校准微升移液器</td><td>吉尔森</td><td></td></tr><tr><td>磁力搅拌器，搅拌棒。 </td><td></td><td></td></tr><tr><td>玻璃烧杯（100，50，20毫升）。 </td><td></td><td></td></tr><tr><td>容量瓶（2毫升）。 </td><td></td><td></td></tr><tr><td> 0.2和2毫升Eppendorf管。 </td><td> Eppendorf公司</td><td></td></tr><tr><td>猎鹰™管各5ml15毫升。 </td><td>鹘</td><td></td></tr><tr><td>实验室旋涡混合器</td><td></td><td>不要使用旋涡混合器重悬磁珠涂HT2 - KLH或联系，以避免proteic部分变性与免疫链</td></tr><tr><td>实验室烘箱或恒温室</td><td></td><td>选择一个烤箱能够保持温度为37 ± 3 ° C。 </td></tr></table>

elime (enzyme linked immuno magnetic electrochemical) method for mycotoxin detection

免疫分析是一种有效的替代更昂贵和时间耗费定量高效液相色谱法或气相色谱1，2为食品类商品中的有害霉菌毒素的筛选检测方法。在这个协议中，我们将展示如何编造并询问电化学竞争性酶联酶联免疫检测，基于磁珠免疫组织化学链 3和丝网印刷电极感应平台的坚实支持。 我们的方法旨在确定总额的HT - 2和T - 2毒素，霉菌毒素属于trichothecenes家庭和 4对人类健康的极大关注。使用了对HT - 2和T - 2的100％的交叉反应的抗体克隆允许同时检测两种毒素具有相似的灵敏度5。 我们检测的第一步是涂层的步骤，我们固定在磁珠表面HT2 - KLH偶联物毒素。后阻塞的一步，要避免非特异性吸收，另外一种单克隆抗体，使固定HT - 2和自由的HT - 2或T - 2样品中或溶解在一个标准的解决方案之间的竞争。 在年底的竞争一步，单克隆抗体的数量挂钩的固定HT - 2将样品溶液中的毒素量成反比。 碱性磷酸酶（AP）标记二抗是用来揭示之间的特异性抗体和固定的HT - 2的结合。最终的测量步骤是下降磁珠悬液等分的，对应一个特定的样品/标准的解决方案，一个丝网印刷工作电极表面上;磁珠固定和集中划归正是磁铁丝网印刷电极。经过两分钟的孵化磁珠和一个AP的基板之间，酶产品检测微分脉冲伏安法（DPV）使用便携式仪器（PalmSens）也能够启动几秒钟的时间间隔内自动八个测量。

Watch this Scientific Journal Video about ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection at JoVE.com

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

检测trichothecenes（霉菌毒素对人类健康的关注），使用一种新开发的筛查方法的基础上有竞争力的免疫组织化学方法和最后的电化学检测的一个协议证明。

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

elime-enzyme-linked-immuno-magnetic-electrochemical-method-for

Research

JoVE Journal

Biology

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

14.1K 次观看.  University of Rome, Tor Vergata. 检测trichothecenes（霉菌毒素对人类健康的关注），使用一种新开发的筛查方法的基础上有竞争力的免疫组织化学方法和最后的电化学检测的一个协议证明。

视频: ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

科研

生物学

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

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

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

利用磁调制生物传感系统的生物检测的快速均质检测

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

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

使用洗涤剂和酶无酶方法从整个组织进行核分离

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.