这项工作是由德国联邦教育与研究部的赠款支持（项目BiGRUDI [毕ologische摹efahrenlagen：R isikobewertung，ü ltraschnelle ð etektion 和我dentifizierung冯bioterroristisch relevanten Agenzien，13N9594]） 。我们感谢B. Diemar（Charité Universitätsmedizin柏林，研究所献给Pharmakologie）优秀的技术援助。

1。通过PEG沉淀提取协议 - 卵黄图1给出了一个IgY的提取过程示意图（见表2）。该协议最初是在波尔森等 。 1980年。 使用乳胶手套，应执行的所有步骤。 <ol><li>仔细蛋壳破裂，蛋黄转移到一个“蛋黄勺”，以尽可能多的蛋白尽可能删除。 </li><li>蛋黄是转移到滤纸和冷轧删除余下的蛋清，蛋黄皮肤被切断与柳叶刀或类似工具（枪头）。倒入50毫升管蛋黄和蛋量（V1），注册。 </li><li>两次蛋黄体积的PBS混合蛋黄（ΣV1+ V2），其后为3.5％PEG 6000总量（克，煤粉）和振荡，其次是在一个滚动的混频器滚动10分钟。这一步分离提取过程分两个阶段进行停牌。一个阶段，包括“蛋黄固体和脂肪类物质”（原报价波尔森等人，1980年）和水样含有卵黄抗体和其他蛋白质的阶段。 </li><li>离心管是在4 ° C为20分钟（每分钟10000转13000 XG，贺利氏Multifuge 3SR +固定角度转子）。上清（V3）浇通过折叠滤芯，并转移到一个新的管。 </li><li>添加8.5％聚乙二醇6000克（根据新卷）管，振荡并在第3步中的滚动搅拌机推出。 </li><li>重复第4步的差异，被丢弃上清。 </li><li>仔细颗粒溶解在1 mL PBS玻璃棍和vortexer。 PBS是一个10毫升的最终卷（V4）。解决的办法是混有12％的PEG 6000（W / V，1.2克），并在第3步（旋涡，滚动混频器）治疗。 </li><li>重复步骤6和800μLPBS溶解沉淀仔细（玻璃粘和涡）。等待气泡消失，然后转移（​​吸管）洗肾胶囊提取。 400μLPBS冲洗管和添加量的透析设备（V5）。 （见透析设备和膜的制备附录。） </li><li>提取物是夜间透析超过0.1％的盐水（1600毫升），通过磁力搅拌器轻轻搅拌。第二天早晨，用PBS代替生理盐水，再过三个小时的透析。 </li><li>此后IgY的提取物是从透析胶囊拉吸管，并转移至2ml管。最终体积约2毫升（V6发动机）。 </li><li>光度测量的样品的蛋白质含量（毫克/毫升）是在280 nm（用PBS稀释1:50），根据朗伯 - 比尔定律计算为IgY的消光系数为1.33（见图2），图。 4显示了该制剂的质量（纯度和回收率都在80％左右）。 </li><li>这是最好存储在样品分装于-20℃（不冻结的样品在-70 ° C）。 </li><li>在朱庇特的协议描述<a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a>的最后准备工作的质量是由简单的SDS - PAGE分析</li></ol> 2。附录使用前透析袋，必须根据制造商的建议，准备以下列方式： <ol><li> 20透析袋切割件30厘米，在一个2000毫升的玻璃烧杯。 </li><li> 1750毫升5毫米EDTA解。 </li><li>一个玻璃漏斗放在袋以上，以确保袋是由EDTA溶液。解决的方法是加热煮沸5分钟（热板）。解决的办法是转自袋是用蒸馏水洗涤三次。 </li><li>再次1,750毫升EDTA的解决方案，补充说，煮沸5 min，用蒸馏水洗涤三次以上。 </li><li>最后，透析袋煮沸蒸馏水10分钟，并保存在4 ° C。消毒镊子取出透析袋。 </li></ol> 3。代表性的成果<img src="/files/ftp_upload/3084/3084fig1.jpg" alt="图1" /> 图1。卵黄提取的示意图，根据聚乙二醇沉淀波尔森等人的原始描述。 1980年， <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">要查看一个较大的图像，请点击这里</a> 。 <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="图2" /> 图2的年龄依赖性的免疫母鸡的鸡蛋蛋黄中总IgY的发展。是每周平均总IgY的/蛋和SD（N = 4蛋鸡）。从[Pauly 等人，2009年]）。 <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="图3" /> 图3。拉依吴四只蛋鸡，不同的抗原免疫能力监测。箭头指示免疫日期（从[Pauly 等人，2009年]）。 <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">查看大图请点击这里。</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="图4" /> 图4。还原条件下个人IgY的准备工作，从不同的鸡蛋聚丙烯酰胺凝胶电泳 巷1 - 2巷 - IgY的标准，巷3-5 - 不同IgY的样品分子量标记，准备在协议中所述。这些最后的准备，根据该协议的步骤10。慧聪 - 重链，液相色谱 - 光chaines，？ -相应的分子量约35 kDa的（可能卵黄二，易制毒化学的C -末端片段，[Klimentzou等，2006]）的细微杂质。 <table border="1"><tbody><tr><td>&nbsp;</td><td> 鸡21（抗原A） </td><td> 鸡22（抗原A） </td><td> 鸡19（抗原乙） </td><td> 鸡23（抗原彗星） </td></tr><tr><td>产蛋数（总IGY）1，2年期</td><td> 625（38克） </td><td> 626（38克） </td><td> 545（33克） </td><td> 608（37克） </td></tr><tr><td>产蛋数（总IgY的），第一年</td><td> 345（20克） </td><td> 304（16克） </td><td> 326（18克） </td><td> 308（17日克） </td></tr><tr><td>产蛋数（总IgY的），第二年</td><td> 280（18克） </td><td> 322（21克） </td><td> 219（15克） </td><td> 300（20克） </td></tr><tr><td> ％最大。可能产蛋数（第一年）2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> ％最大。可能产蛋数（第二年） </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td>加工蛋</td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 IgY的总额是60毫克每鸡蛋的蛋白质，根据图2中的数据的平均值乘以一个鸡蛋的数量计算。 2最大。根据饲养员的信息可能每年产蛋数为325。 表1。两年的统计数据具有不同的抗原免疫鸡的产蛋能力。铺设与不同的抗原，以及在两年IgY的结局后的免疫能力，最多四只蛋鸡％（参见图3）， 保利等人，2009年] ）。 <table border="1"><tbody><tr><td> 蛋NR。 </td><td> 母鸡NR。布设日期 </td><td> V1 [ML]蛋黄 </td><td> V2 [ML] PBS </td><td> 1。聚乙二醇PREC。 [G] </td><td> VOL [ML]后 </td><td> 2。聚乙二醇PREC。 [G] </td><td> 弹丸 </td><td> 3。聚乙二醇PREC。 [G] </td><td> 弹丸 </td><td> VOL [毫升] </td><td> 总蛋白毫克/毫升 </td><td> 矫正总蛋白毫克/毫升 </td></tr><tr><td></td><td></td><td></td><td> 2xV1ΣV1+ V2 </td><td> 3.5％W / V]ΣV1+ V2 </td><td> PREC。centrif。和过滤的V3 </td><td> 8,5％[W / V] V3 </td><td>溶解于毫升PBS V4 </td><td> 12％[W / V] V4 </td><td>溶于毫升PBS V5 </td><td>经过透析对PBS V6 </td><td> A280/ml </td><td> A280/ml：1.33 </td></tr><tr><td> 1 </td><td> H293 / 22.10。 </td><td> 15 </td><td> 30 45 </td><td> 1.58 </td><td> 31 </td><td> 2.63 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 35.0 </td><td> 26.3 </td></tr><tr><td> 2 </td><td> H293 / 23.10。 </td><td> 13 </td><td> 26 39 </td><td> 1.37 </td><td> 25 </td><td> 2.12 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 28.8 </td><td> 21.6 </td></tr></tbody></table> 表2 PEG沉淀模范协议。

<ol>
	<li>Akita, E. M., Nakai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+purification+methods+for+the+production+of+immunoglobulins+from+eggs+laid+by+hens+immunized+with+an+enterotoxigenic+E.+coli+strain.">Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.</a> J. Immunol. Methods. 160, 207-214 (1993).</li><li>De Meulenaer, B., Huyghebaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+chicken+egg+yolk+immunoglobulins:+a+review.">Isolation and purification of chicken egg yolk immunoglobulins: a review.</a> Food Agricult. Immunol. 13, 275-288 (2001).</li><li>Klimentzou, P., Paravatou-Petsotas, M., Zikos, C. h., Beck, A., Skopeliti, M., Czarnecki, J., Tsitsilonis, O., Voelter, W., Livaniou, E., Evangelatos, G. P. <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+immunochemical+evaluation+of+antibodies+Y+for+the+poorly,+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly, immunogenic polypeptide prothymosin alpha.</a> Peptides. 27, 183-193 (2006).</li><li>Pauly, D., Dorner, M., Zhang, X., Hlinak, A., Dorner, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+laying+capacity,+immunoglobulin+Y+concentration,+and+antibody+titer+development+in+chickens+immunized+with+ricin+and+botulinum+toxins+over+a+two-year+period.">Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.</a> Poultry Science. 88, 281-290 (2009).</li><li>Polson, A., von Wechmar, M. B., van Regenmortel, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+viral+IgY+antibodies+from+yolks+of+immunized+hens.">Isolation of viral IgY antibodies from yolks of immunized hens.</a> Immunol. Commun. 9, 475-493 (1980).</li><li>Schade, R., Behn, I., Erhard, M., Hlinak, A., Staak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chicken egg yolk antibodies, production and application : IgY-Technology. , (2001).</li><li>Schade, R., Calzado, E. G., Sarmiento, R., Chacana, P. A., Porankiewicz-Asplund, J., Terzolo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+egg+yolk+antibodies+(IgY-technology):+a+review+of+progress+in+production+and+use+in+research+and+human+and+veterinary+medicine.">Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.</a> Altern. Lab Anim. 33, 129-154 (2005).</li><li>Witkowski, P. T., Bourquain, D. R., Hohn, O., Schade, R., Nitsche, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun-supported+DNA+immunisation+of+chicken+for+straightforward+production+of+poxvirus-specifiv+IgY+antibodies.">Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specifiv IgY antibodies.</a> J. Immunol. Methods. 341, 146-153 (2009).</li></ol>

动物实验Landesamt毛皮Gesundheit和Soziales柏林（H 0069/03）所规定的准则和法规的规定执行。

选择一个合适的IgY的提纯方法是影响净化（实验室或工业），成本效益，技术（实验室设备），和对环境的影响（废物管理）的规模。德Meulenaer Huyghebaert（2001年，审查也看到SCHADE 等 ，2005）详细审查各种IgY的提取方法。在一般情况下，这些方法可分为三个主要群体： <ol><li> 沉淀法：涉及铵或硫酸钠，聚乙二醇（PEG），辛酸和caragenean。 </li><li> 色谱方法：thiophylic相互作用层析，疏水层析，亲和层析，离子交换层析，凝胶过滤色谱。 </li><li> 超滤 。 </li></ol> IgY的制备纯度可提高相结合的方法，例如，PEG沉淀可与亲和层析结合。在某些情况下，取决于最终的应用程序，IgY的水提取物足以达到良好的效果（秋田县井1993年）。根据我们的经验，IgY的样本，我们获得PEG沉淀在很多不同的免疫检测工作非常出色。通过规范的PEG沉淀法，一个技术助理是能够处理约84鸡蛋每星期，这相当于4至7克，每周的总卵黄金额。一个简单的计算：一个蛋鸡是一种抗原免疫接种。经过几次提高免疫接种母鸡的生产与1:50,000滴度的特异性抗体（AB）在为期30天。 30鸡蛋× 2毫升卵黄提取物（见协议和图3），相当于总卵黄60毫升，这反过来又对应AB - 3000升的稀释（一斗的容量为10升），这意味着有3000升AB的解决方案，300桶，可填补。因此，在数量如此巨大的AB -的存储稳定的质量和特异性抗体池，它是没有问题的。这种技术减少了充电/很多的可变性，否则在多特异性抗体观察。 尽管数量方面，卵黄抗体与哺乳动物IgG抗体相比有更多的优势：没有激活哺乳动物补体系统，没有交叉反应HAMA（人抗鼠抗体），类风湿因子或人类的血型抗原（heteroagglutinins缺乏）。 卵黄抗体的一个突出的优点是所谓的进化距离。亲缘距离是经常描述的哺乳动物和鸡的抗体特异性之间差异的原因，即使在相同的免疫。除了哺乳动物和禽流感的免疫系统本身之间的差异，在进化发展的差异，这两个动物类AB不同的特殊性。几位作者报道，鸡往往会产生ABS对系统发育上高度保守的哺乳动物蛋白或多肽更有效地比兔（审查SCHADE等A. 2005）。因此，可以保持一个保守的抗原“蒙面”兔的免疫系统，从而导致只有微弱或“沉默”回应。此外，如果鸡兔是哺乳动物具有相​​同的抗原免疫的，往往是鸡与AB -特异性，很少能在兔子取得回应。动物福利方面是重要的，因为是AB从蛋黄中提取的非侵入性。结合基因枪（质粒）免疫IgY的生产是完全非侵入（Witkowski 等 2009）。 总之，母鸡和PEG沉淀IgY的提取生产的AB是非常符合成本效益和高度特异性的与稳定的滴度AB以1:1,000,000结果。由于鸟类和哺乳动物之间的进化距离，鸡能够产生对特定的高度保守的哺乳动物抗原抗体，如兔。 IgY的技术获得数额特别巨大的AB，打开门也使用在人类医学和兽医学卵黄抗体治疗/预防的目的。

<ol><li>透析袋Visking，27/32型，切断-14 KD，卡尔罗斯公司，德国卡尔斯鲁厄</li><li>猎鹰管蓝最大，50毫升聚丙烯锥形管，碧迪医学实验，富兰克林湖，美国纽约</li><li>折叠滤芯百万615 ¼，∅150毫米，卡尔罗斯公司，卡尔斯鲁厄，德国Heraeus </li><li>磁力搅拌器，Variomag单声道，H + P实验室技术股份公司，Oberschleißheim，德国</li><li>微透析胶囊5,0毫升，卡尔罗斯公司，德国卡尔斯鲁厄</li><li> Multifuge 3SR +，Thermo Scientific的Langenselbold，德国</li><li>光度计UV - 160A，日本岛津德国公司，德国杜伊斯堡</li><li>聚乙二醇6000，Rotipuran药典，卡尔罗斯公司，德国卡尔斯鲁厄</li><li>预混PBS缓冲10X，罗氏诊断有限公司，德国曼海姆</li><li>滚筒搅拌机SRT2，司徒，英国斯塔福德郡， </li><li> Vibrofix VF2，Janke Kunkel，德国IKA，实验室技术，德国，诗道芬i.Br. </li><li>蛋黄勺，Fackelmann（houshold效果），德国</li></ol>

igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation

母鸡可以通过IM疫苗接种（ 胸肌 ，左，右，注射量为0.5-1.0毫升）的手段，或通过质粒基因枪免疫接种。依赖于抗原的免疫原性，高的抗体滴度（高达1:100000 - 1:1,000,000）可以达到后，只有一个或3 - 4提高免疫接种。通常，一只母鸡下蛋持续约72周，随后铺设能力下降。这个协议描述手段（PEG.波尔森等人 ，1980年）的降水过程，从蛋黄中总IgY的提取。该方法包括两个重要的步骤。第一个是清除血脂，第二个是从第一个步骤上清总IgY的降水。反对的缓冲液（通常PBS）透析后的IgY的提取物可以储存在-20 ° C了一年多。提取的纯度在80％左右，每个鸡蛋的总IgY的不同从40-80毫克，依赖于蛋鸡年龄。总IgY的母鸡的年龄大约为40毫克/蛋高达80毫克/蛋（关于PEG沉淀）含量的增加。铺设了母鸡每年的容量大约为325鸡蛋。这意味着潜在总收成的20克总卵黄/年的平均卵黄含量60毫克的总IgY的/蛋（见表1）的基础上。

Watch this Scientific Journal Video about IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation at JoVE.com

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

这个协议描述，尤其是从蛋黄IgY的提取由聚乙二醇沉淀的手段，并给出了有关IgY的技术信息。

IgY的技术：从蛋黄鸡抗体提取聚乙二醇（PEG）降水量

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

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IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

64.1K 次观看.  Robert Koch-Institute. 这个协议描述，尤其是从蛋黄IgY的提取由聚乙二醇沉淀的手段，并给出了有关IgY的技术信息。

视频: IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

科研

免疫与感染

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

通过横向流技术检测在血液系统恶性肿瘤患者侵袭性肺曲菌病

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...