We appreciate the colleagues in Taiwan Cetacean Society, Marine Biology and Cetacean Research Center of National Cheng Kung Univerisy, Farglory Ocean Park, and Animal Disease Diagnostic Center of National Chiayi Universiy for sample collection. This project was funded by grant to WCY from the Council of Agriculture of Taiwan (100AS-02.1-FB99).

道德守则:由嘉义大学，批准编号的机构动物护理和使用委员会（IACUC）按照国际准则进行研究并批准:99022.鲸类动物样品的使用是由台湾农业委员会（研究许可证许可100M-02.1-C-99）。 1.肌肉样品制备和SDS-PAGE 注:从23种肌肉样本，包括16种海洋哺乳动物，5种陆栖哺乳类动物，金枪鱼和鸡肉在这项研究中（ 表1）使用。鲸类动物的肌肉样本来自个人搁浅，误捕渔业，和没收所得。兔，鼠，狗和鸡肌肉组织从嘉义大学的动物疫病诊断中心获得。牛肉，猪肉，羊肉，鱼和金枪鱼的样品从当地超市购买。斑海豹（ 斑海豹的肌肉样本</eM&gt;）由远雄海洋公园提供。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）来可溶性蛋白与肌肉样品在不同的分子量分离。 <ol><li>在-20°C，直到使用存储所有样本。 </li><li>在-20℃的预冷砂浆。然后把3克冰冻肌肉样品进去。 </li><li>均质使用组织匀浆10毫升凉爽磷酸盐缓冲盐水（PBS）中的样本。 </li><li>离心匀浆样品以10,000 xg离心在4℃下10分钟。收集上清液并储存于-20℃直到使用。 </li><li>制备5毫升5％浓缩胶（3.07毫升蒸馏水，1.25毫升液pH 6.8，625微升40％的丙烯酰胺，50微升10％过硫酸铵（APS）的，和5μl四甲基乙二胺的4倍上凝胶缓冲液（ TEMED））和10毫升15％分离胶（3.65毫升蒸馏水，2.5毫升4倍的pH值8.8下的凝胶缓冲液，3.75毫升40％的丙烯酰胺，加入100μl的10％APS，和4微升TEMED的）。 <ol><li>在电泳单元，倒入堆叠上分离凝胶（15％丙烯酰胺），后者已凝固后的顶部凝胶（5％丙烯酰胺）。插入在层叠凝胶的凝胶梳子。 </li></ol></li><li>按照下列条件进行PAGE:初始运行条件:100伏，20分钟，并最终条件:120伏，40分钟。 </li><li>染色在室温下用考马斯亮蓝将凝胶进行30分钟，直到凝胶的颜色均匀的蓝色。 注:染色完成后的凝胶不再是在染料溶液中可见。 </li><li>在室温下用乙酸溶液（10％）1小时脱色它。乐队将开始出现。继续在4℃过夜，脱色直到背景是清楚的。 </li></ol> 2.多肽合成和单克隆抗体生产<ol><li>从GenBank中检索兆的氨基酸序列包括金枪鱼，鸡，鸵鸟，家养哺乳动物，密封和18种鲸类（ 表2）。 </li><li>使用对齐适当的软件16的序列: <ol><li>通过选择对齐启动对齐浏览器:编辑/编译启动栏上的对齐 ;选择创建新对齐 ，然后单击确定 。将出现一个对话框，询问"你建立一个DNA或蛋白质序列比对？" </li><li>点击标有" 蛋白质 "按钮;选择数据:打开:检索文件 ，并选择序列文件序列 ;选择编辑:全选菜单命令，选择所有网站在数据创建多序列比对每一个设置序列。 </li><li>选择路线:由ClustalW比从主菜单中对齐使用的ClustalW算法选择的序列数据对齐 ;选择"BLOSUM"作为蛋白质权重矩阵，然后点击确定按钮。 </li></ol></li><li>分析序列比对: <ol><li>专注于5抗原反应SITES 17:站点1（AKVEADVA，15-22），现场2（KASEDLK，56-62），现场3（ATKHKI，94-99），现场4（HVLHSRH，113-119），与本站5（KYKELGY，145- 151），并发现鲸类动物中是保守的片段。 *（星号;在对齐共识符号（协议2.2.3））表明其中有一个单一的，完全保守残基的位置。在鲸类下面的保守片段发现:序列KASEDLKKHG（包括​​站2）和序列HVLHSRHP（包括站4）。 </li></ol></li><li>根据序列分析，并用卵清蛋白蛋白（OVA）作为使用商业服务载体蛋白质缀合物合成候选序列片段。 <ol><li>添加疏水性氨基酸（ 例如 ，甲硫氨酸），以抗原反应部位的N-末端，用于防止所述肽从分解（ 例如 ，M-KASEDLKKHG）。 </li><li>延长的抗原反应部位的C末端的核心抗原性位点暴露于免疫细胞。此外，conju栅极通过加入半胱氨酸（Cys，C）与C末端以OVA肽（ 例如 ，M-KASEDLKKHG-NTVL-C）。 </li></ol></li><li>乳化完全弗氏佐剂免疫原1或弗氏不完全佐剂对免疫原2在PBS等体积各合成肽（3毫升，最终浓度30-50微克/ 100微升）。 </li><li>接种免疫原1（0.1毫克）皮下注射到各5只雌性BALB / c小鼠。 </li><li>在两周的时间间隔执行皮下加强注射用免疫原2五次，每次增压之前收集来自小鼠尾部剪裁取样测试血清。 </li><li>确定用于第一次筛选的血清滴度。 <ol><li>溶解100微克游离肽在25毫升反应缓冲液和试样50微升溶液中加入96孔板的每个孔中。加10微升偶联试剂溶液导入各孔中，并混合板。将培养板在室温下2小时。 </li><li>除去孔中的内容，用蒸馏水洗各孔3次，并通过加入200微升的封闭溶液的遮挡板。孵育在室温下1小时。移除的内容，并用蒸馏水洗涤各孔3次。 </li><li>用于确定第一轮筛选血清效价进行间接ELISA（协议5.1-5.7）。选择与脾集最高价（最高光密度）鼠标。 </li></ol></li><li>脾收集前三天，接种0.1毫克免疫原1的皮下到小鼠呈现最高滴度。 </li><li>收集来自所选择的免疫的小鼠和保险丝与鼠骨髓瘤细胞F0（SP2 / 0-AG14）的脾细胞，以获得杂交瘤细胞单抗生成18。 </li><li>融合后14天内，通过筛选倾向于使用间接ELISA免费合成肽的杂交瘤上清液（协议5.1-5.7）的反应选择阳性克隆。 </li><li>稀释细胞以适当数量，每孔为maximizING仅包含一个单一的克隆（稀释克隆）的孔的比例。 <ol><li>添加100μl的细胞培养基中，除了该空孔A1中的96孔板的所有孔中。 </li><li>加入200微升细胞悬浮液至A1孔。然后迅速从A1到B1转移100微升，并轻轻吹打混合。重复这些1:2稀释了整个列，然后丢弃从H1 100微升，使其具有相同的体积，它上面的孔中结束。 </li><li>额外的100微升培养基中加入1列有一个8通道微量。然后快速地从每个孔中在第1列使用相同的移液管转移100μl到那些在第2栏，并轻轻吹打混匀。 </li><li>使用同样的技巧，重复这些1:2稀释整个板块。丢弃从每个孔中加入100μl在最后一列。 </li><li>通过加入到每个100微升培养基以及将所有孔中以200微升的最终体积。解放军孵育德在加湿的 CO 2培养箱中不受干扰在37℃。 </li><li>检查每口井，并标记包含只是一个单一的殖民地所有的井。开展两个或两个以上clonings直到包含单克隆井&gt; 90％是阳性抗体产生。 </li></ol></li><li>屏幕Western blot和斑点杂交（协议3.1-4.6）的克隆。然后，从水井到大型船舶的亚文化群体扩大获得单克隆抗体的细胞。一般每个克隆被转移到一个单一的井在12或24孔板中。 </li><li>测量单克隆抗体和牛，羊，猪，狗，兔，金枪鱼，鸡肉，密封肌肉提取物，并通过免疫印迹和斑点杂交（协议3.1-4.6）四个具有代表性的鲸类之间的亲和力。 </li><li>接种选定杂交瘤细胞（最多至3×10 6）腹膜内入小鼠以诱导腹水。腹部肿胀通常是明显在7-10天后注射杂交瘤细胞。收集流体使用皮下注射针（小于20号）。 </li><li>离心机腹水液（10,000×g离心10分钟）以去除细胞和碎片。通过0.45微米的过滤器过滤。添加1〜20毫升样品，15毫升结合缓冲液和3至5ml洗脱缓冲液进G蛋白Sepharose柱。收集含有从小鼠腹水纯化抗体的洗脱级分。 </li><li>确定由抗体分型试剂盒使用制造商的说明将抗体同种型。 </li></ol> 3.免疫印迹<ol><li>制备2×上样缓冲液通过混合950微升2×Laemmli样品缓冲液中以50μl的BME的含有β巯基乙醇（BME）。使用多克隆兔抗人兆抗体时5（家畜和金枪鱼），以及:淡化肌肉上清液（协议1.1-1.4）的加样缓冲液以合适的比例以获得良好的信号:1:50（鲸类和密封）和1- 1:1（猪，兔，鸡和金枪鱼），1:5（牛，羊和狗）和1:25（鲸类和盖章）时，ntibody是从杂交瘤上清液。 </li><li>在95℃下5分钟热样品。负载样品到SDS-PAGE凝胶（4％丙烯酰胺堆叠和12％丙烯酰胺分离）与分子量标记物沿着所述孔中。在50运行5分钟的凝胶V，那么增加的电压为150V以完成运行在约1小时。 </li><li>放置在1×转移缓冲液的凝胶15分钟。分离的蛋白转移到硝酸纤维素（NC）膜后，他们用PAGE分离。传输可以在100V进行60-90分钟。 </li><li>制备阻断溶液:含有1×PBS中的0.1％吐温20，用5％的脱脂奶粉。在25毫升阻断在室温下溶液1小时的阻断NC膜。洗涤三次，每次5分钟，用吐温20（PBST）15毫升磷酸盐缓冲盐水。 </li><li>孵育在10毫升抗体稀释缓冲液在4℃下用5％封闭溶液稀释过夜膜与第一抗体（腹水或杂交瘤上清液）。 </li><li>洗MEMBRANË三次PBST再擦去过多的抗体。 </li><li>在温和搅拌，在室温下1小时，阻断溶液1250:在1孵育用碱性磷酸酶缀合的山羊抗小鼠IgG的膜。 </li><li>再次洗膜，并在5-溴-4-氯-3-吲哚基磷酸盐/对 - 氮蓝四唑氯化物孵育它（BCIP / NBT）磷酸酶底物混合物为10至20分钟，直到显色。 </li><li>通过在蒸馏水几个变化洗膜终止反应。 </li></ol> 4.点杂交<ol><li>稀释肌肉上清液（协议1.1-1.4）在PBST 5％牛血清白蛋白（BSA）以适当的比例获得良好的信号:1:5的家畜和金枪鱼和1:25鲸类和密封。现货5微升样品到膜上。最小的区域，该解决方案通过采用缓慢它渗透（一般3-4毫米直径）。 </li><li>在室温（干燥膜如&lt;在30-60分钟的层流/ em&gt;的），用封闭在培养皿溶液在室温下1小时阻止它，并用PBST清洗。 </li><li>孵育初级抗体的膜（单抗从杂交瘤上清液在1:10,000或腹水流体在1:100 000 5％封闭溶液稀释的）在室温下1小时。 </li><li>使用PBST洗涤膜3次，每次5分钟（1碱性磷酸酶缀合的山羊抗小鼠IgG:1.250，在5％封闭溶液）以除去过量的抗体，然后用第二抗体孵育它在室温下1小时。 </li><li>再次洗膜，并在10至20分钟的BCIP / NBT磷酸酶底物混合物，直至显色孵化它。 </li><li>通过在蒸馏水几个变化洗膜终止反应。 </li></ol> 5.间接ELISA <ol><li>制备洗涤缓冲液（0.002 1M咪唑缓冲盐水0.02％吐温20）。洗净板洗涤3次每个接下来的步骤（协议5.2-5.5）之间的缓冲。 </li><li>准备1:25稀释涂料缓冲肌肉上清液（协议1.1-1.4）的。涂层的96孔ELISA板用100μl稀释的上清液在4℃下过夜，用在室温下1小时封闭缓冲液（在PBS中的1％BSA）阻止它。 </li><li>制备1:2000稀释，用稀缓冲纯化mAb的和稀释的mAb添加100μl的各孔中。将培养板在室温下1小时。 </li><li>加入山羊抗小鼠IgG缀合的辣根过氧化物酶（1:200稀释在稀释缓冲液中）进行进一步孵育。 </li><li>添加过氧化物酶底物至每孔（100μl/孔）并孵育10至15分钟。 </li><li>停止时通过彩色显影观察到过氧化物酶终止溶液（100μl/孔）的酶反应。 </li><li>读在使用微板分光光度计450nm处的光密度。 </li></ol> 6.制备胶体金标记的单克隆抗体的</p&gt; 注意:胶体金溶液中，混合物的颜色应始终为红色。当黑色的沉淀物发现调节pH，单克隆抗体浓度，离心机转速。步骤6.1和6.2的优化步骤。 <ol><li>添加纯化的检测的mAb（50微克/毫升，3微升）至100μl随着pH值从5-9不同的胶态金溶液。该保持红色两小时的最小pH值被视为最适PH值。注意:在此研究中，0.1M碳酸钾用于调整胶体金（40纳米）溶液至pH 8.0（最适pH）。 </li><li>至100微升胶体金溶液的加入各种数量的纯化检测mAb的（500微克/毫升，1-20微升）在pH 8.0。注意:在此研究中的最佳浓度为6微克/毫升（无黑色沉淀）。 </li><li>根据上述结果，添加的纯化检测单克隆抗体滴加至10ml胶体金溶液的60微克。轻轻乳化混合物在室温下10分钟。广告D 2毫升在PBS中的5％BSA的溶液（pH 7.4）至该混合物中并在室温下轻轻地乳化15分钟，以减少背景干扰。 </li><li>离心该混合物以10,000 xg离心在4℃下30分钟。 </li><li>小心地取出与未缀合的抗体上清液并暂停将所得丸粒在4毫升的PBST含有1％BSA和0.1％吐温20，并重复离心和悬浮液数次。 </li><li>暂停的最终沉淀在1ml PBST并储存于4℃直至使用。 </li></ol> 7.免疫带建设注意: 图1显示了免疫条设计。制备和组装&lt;以延长储存寿命（20％相对湿度（1年）在低湿度的实验室环境条件下的条带）&gt;。垫和膜的尺寸为:结合垫300毫米×10毫米，吸收垫300毫米×24毫米，样品垫300毫米×24毫米，NC膜300毫米×25毫米，粘贴板300毫米×80毫米。 <ol><li>添加来自步骤6.6的胶体金标记的单克隆抗体溶液用微量至饱和的偶联物垫，然后组装前在37℃下使其干燥1小时。 </li><li>在控制区分配抗原特异性捕获在测试区单克隆抗体（500微克/毫升）和兔抗小鼠IgG（500微克/毫升），用于使用吸液管或免疫条打印机NC膜上检测的mAb。这两个区域之间保持距离（&gt; 5mm）的，以避免干扰。 </li><li>粘贴复合垫，吸水垫和NC膜与双面胶带粘贴板。 注意:通过约2mm重叠在NC膜的每一侧的垫。不适当带建设将导致不完全的测试。 </li><li>将在结合垫样品垫（2毫米），将其粘贴在纸板。 </li><li>创建具有切纸机6毫米宽的条带。包在铝箔袋干燥剂的条带，且他们在4℃储存直至使用。 </li></ol> 8.交叉反应试验<ol><li>均匀0.03克原肌样品用竹签或研磨棒1ml的PBS（含有0.1％BSA）在1.5毫升离心管中。 </li><li>由端相对的测试区域保持条和样品垫部浸入试样5-10分钟，并直接观察结果。 <ol><li>可选:收集500微升上层清液，并转移到一个新的离心管中。 注意:如果当带材被直接浸透到上清液中的信号并不明显建议此步骤。 </li></ol></li><li>一式三份测试各种肌肉样品。 注:在这里，我们测试的金枪鱼，鸡肉，密封，15种鲸类动物5种陆生哺乳动物。 </li><li>有五个独立的监察员8.3重复五次， 即在总使用575条。 </li></ol>

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Technol. 39, 5703-5708 (2005).</li><li>Tryland, M., 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=Human+pathogens+in+marine+mammal+meat.">Human pathogens in marine mammal meat.</a> Norwegian Scientific Committee for Food Safety (VKM). , (2011).</li><li>Matsunaga, T., 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=A+quick+and+simple+method+for+the+identification+of+meat+species+and+meat+products+by+PCR+assay.">A quick and simple method for the identification of meat species and meat products by PCR assay.</a> Meat Sci. 51, 143-148 (1999).</li><li>Dalebout, M. L., Helden, A. V., van Waerebeek, K., Baker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+identification+of+southern+hemisphere+beaked+whales+(Cetacea:+Ziphiidae).">Molecular genetic identification of southern hemisphere beaked whales (Cetacea: Ziphiidae).</a> Mol. Ecol. 7, 687-694 (1998).</li><li>Dalmasso, 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=A+multiplex+PCR+assay+for+the+identification+of+animal+species+in+feedstuffs.">A multiplex PCR assay for the identification of animal species in feedstuffs.</a> Mol. Cell. Probes. 18, 81-87 (2004).</li><li>Kesmen, Z., Sahin, F., Yetim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+assay+for+the+identification+of+animal+species+in+cooked+sausages.">PCR assay for the identification of animal species in cooked sausages.</a> Meat Sci. 77, 649-653 (2007).</li><li>Liu, L., Chen, F. -. C., Dorsey, J. L., Hsieh, Y. -. H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Monoclonal+Antibody-based+Sandwich+ELISA+for+the+Detection+of+Porcine+Skeletal+Muscle+in+Meat+and+Feed+Products.">Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products.</a> J. Food Sci. 71, 1-6 (2006).</li><li>Kotoura, S., 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=A+Sandwich+ELISA+for+the+Determination+of+Beef+Meat+Content+in+Processed+Foods.">A Sandwich ELISA for the Determination of Beef Meat Content in Processed Foods.</a> Food Sci. Techno. Res. 15, 613-618 (2009).</li><li>Hsieh, Y. H., Chen, Y. T., Gajewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody-based+sandwich+ELISA+for+reliable+identification+of+imported+Pangasius+catfish.">Monoclonal antibody-based sandwich ELISA for reliable identification of imported Pangasius catfish.</a> J. Food Sci. 74, 602-607 (2009).</li><li>Ross, H. 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=DNA+surveillance:+web-based+molecular+identification+of+whales,+dolphins,+and+porpoises.">DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.</a> J.Hered. 94, 111-114 (2003).</li><li>Ngom, B., Guo, Y., Wang, X., Bi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+lateral+flow+test+strip+technology+for+detection+of+infectious+agents+and+chemical+contaminants:+a+review.">Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review.</a> Anal. Bioanal. Chem. 397, 1113-1135 (2010).</li><li>Muldoon, M. T., Onisk, D. V., Brown, M. C., Stave, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets+and+methods+for+the+detection+of+processed+animal+proteins+in+animal+feedstuffs.">Targets and methods for the detection of processed animal proteins in animal feedstuffs.</a> Int. J. Food Sci. Technol. 39, 851-861 (2004).</li><li>Rao, Q., Hsieh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+commercial+lateral+flow+feed+test+for+rapid+detection+of+beef+and+sheep+content+in+raw+and+cooked+meats.">Evaluation of a commercial lateral flow feed test for rapid detection of beef and sheep content in raw and cooked meats.</a> Meat Sci. 76, 489-494 (2007).</li><li>Tamura, K., 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=MEGA5:+molecular+evolutionary+genetics+analysis+using+maximum+likelihood,+evolutionary+distance,+and+maximum+parsimony+methods.">MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.</a> Mol. Biol. Evol. 28, 2731-2739 (2011).</li><li>Atassi, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+structure+of+myoglobin:+the+complete+immunochemical+anatomy+of+a+protein+and+conclusions+relating+to+antigenic+structures+of+proteins.">Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins.</a> Immunochemistry. 12, 423-438 (1975).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridoma+technology+for+the+generation+of+monoclonal+antibodies.">Hybridoma technology for the generation of monoclonal antibodies.</a> Methods Mol. Biol. 901, 117-135 (2012).</li><li>Kao, D. J., Hodges, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+a+synthetic+peptide+immunogen+over+a+protein+immunogen+in+the+development+of+an+anti-pilus+vaccine+for+Pseudomonas+aeruginosa.">Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa.</a> Chem. Biol. Drug Des. 74, 33-42 (2009).</li><li>Dolar, M. L., Suarez, P., Ponganis, P. J., Kooyman, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+in+pelagic+small+cetaceans.">Myoglobin in pelagic small cetaceans.</a> J. Exp. Biol. 202, 227-236 (1999).</li><li>Lo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+immune+colloidal+gold+strip+for+cetacean+meat+restraining+illegal+trade+and+consumption:+implications+for+conservation+and+public+health.">Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health.</a> PLoS ONE. 8, e60704 (2013).</li></ol>

The authors declare that they have no competing financial interests.

使用缀合载体蛋白的合成肽相比，它的同源蛋白是显着更有效。对于基于夹层技术，因为单克隆抗体是使用具有已知相对位置的表位开发的，在本研究的两种单克隆抗体不太可能彼此的与靶抗原表位的相互作用来干扰。此外，天然蛋白和合成肽结合物免疫的小鼠的抗体之间的反应性可能比天然蛋白和与天然蛋白19产生的抗体之间的反应性更强。因此，使用合成肽缀合物的建议适当抗肽mAb的...

Historically, cetacean meat has been consumed in many parts of the world and this consumption continues today1. Due to the trophic level of cetaceans, high levels of mercury and other toxins are known to be present in their meat2. Therefore, the consumption of cetacean meat could lead to a health problem not only for high-risk groups such as pregnant women but also for the general population3. Furthermore, the contamination of cetacean meat with zoonotic or potentially zoonotic pathogens can also occur during its processing and storage4. It is difficult even for experienced agents to identify cetacean meats by their appearance alone. Therefore, a reliable scientific method of identification is required to differentiate cetacean meat from other meats. This would help to limit the consumption of cetacean meat.
Current methods of species identification include molecular techniques and immunological methods. Molecular techniques, such as polymerase chain reaction (PCR) and DNA sequencing, can be used to identify samples not only from raw meat5 and decomposed samples6 but also from processed foods such as cooked sausage and feedstuffs7,8. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are commonly applied in food production to detect the meat content of, for example, pork9, beef10 and catfish11. PCR-based DNA analysis for the identification of cetacean meat is available12, and has helped prevent the illegal international trade of cetacean meat in Japan, South Korea, the Philippines, Taiwan, Hong Kong, Russia, Norway, and the United States1. These methods are effective and reliable, but they can take hours or days to complete and involve laborious steps. The identification of cetacean meats is usually based on molecular techniques and there is currently no immunological method available. For regulatory agencies, it is highly desirable to develop a dependable and rapid technique that can be used in the field to identify cetacean meats. 
Immunochromatographic strips are used as detection tools with the advantage of producing rapid result via a simple protocol that is suitable for use in the field. The principles of the immunochromatographic strip and ELISA are very similar, and includes antibodies, antigens and labels. Many different labels such as colloidal gold, carbon and latex have been used in the development of immunochromatographic strips. At present, this method is commonly used for detecting antibiotics, toxin, bacteria and viruses13, but it is rarely used for identifying proteins in meat14,15. Here we propose a lateral-flow chromatographic enzyme immunoassay for rapid detection of cetacean myoglobin (Mb).

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Phosphate buffered saline</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:148px;">J373</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Protein G HP SpinTrap&#160;</td>
			<td>GE&#160;Healthcare</td>
			<td>28-9031-34</td>
			<td>spin column containing Protein G Sepharose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">IsoStrip Mouse Monoclonal Antibody Isotyping Kit</td>
			<td>Roche</td>
			<td>11493027001</td>
			<td>Isotyping Strips, precoated with subclass- and light-chain-specific anti-mouse-Ig antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Trans-Blot</td>
			<td>Bio-Rad</td>
			<td>170-3935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Whatman</td>
			<td>Z613630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody blocker solution&#160;</td>
			<td>LTK BioLaboratories</td>
			<td style="width:148px;"></td>
			<td>To minimize nonspecific binding interactions of nonspecific IgG in the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">BCIP/NBT phosphatase substrate&#160;</td>
			<td>KPL</td>
			<td>50-81-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector HRP Microwell Kit, Anti-Mouse</td>
			<td style="width:183px;">KPL</td>
			<td>54-62-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Immunoplate MaxiSorp ELISA plate</td>
			<td>Thermo&#160;Fisher Scientific</td>
			<td>EW-01928-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiskan&#160;EX ELISA reader&#160;</td>
			<td>Thermo&#160;Electron&#160;Corporation</td>
			<td>51118170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloid gold (40 nm) solution&#160;</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td>40-50 nm is appropriate for immunostrip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Gibco</td>
			<td>15561-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid test immno-strip printer</td>
			<td>REGA biotechnology Inc.</td>
			<td>AGISMART RP-1000&#160;</td>
			<td>Only suited for small scale production of immunostrips for research and development purposes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Strip components (NC membranes, sample pads (#33 glass, S&#38;S), conjugate pads (#16S, S&#38;S) and absorbent pads (CF6, Whatman))</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freund&#8217;s adjuvant and incomplete Freund&#8217;s adjuvant&#160;</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:148px;">F5881, F5506</td>
			<td>Used to produce water-in-oil emulsions of immunogens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED)&#160;</td>
			<td style="width:183px;">Protech</td>
			<td style="width:148px;"></td>
			<td>Gel preparation for SDS-PAGE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie brilliant blue R-250</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610436</td>
			<td>Protein staining in SDS-PAGE gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laemmli sample buffer and &#946;-mercaptoethanol</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610737, 1610710</td>
			<td>Dilute protein samples before loading on SDS-PAGE gels</td>
		</tr>
	</tbody>
</table>

development of a colloidal gold-based immunochromatographic test strip for detection of cetacean myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

单克隆抗体的特点 我们分别开发了两种抗体1单克隆抗体（CGF5H9和CSF1H13）承认两个合成肽（MKASEDLKKHGNTVLC和AIIHVLHSRHPAEFGC），鲸类MB，而这些被用来构建一个三明治式胶体金免疫层析试纸的快速检测鲸类MB的。 图2示出CGF5H9检测鲸类和其他哺乳动物作为在近似17 kDa的预测分子量的单个染色带。该小须鲸（ 鲸acutorostrata）显示出比其他鲸类的?...

Watch this Scientific Journal Video about Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin at JoVE.com

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

对肌红蛋白鲸类检测基于金免疫层析胶体试纸条的研制

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

development-colloidal-gold-based-immunochromatographic-test-strip-for

Research

JoVE Journal

Biology

14.1K 次观看.  National Chiayi University. This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals. 

视频: Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

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 ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

制备大鼠脑组织神经元和神经胶质发展研究总结文化

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

切向流超:A胶体银纳米粒子的大小选择与集中"绿色"方法

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157:H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...