研究合同米格尔SERVET（ISCIII CD14 / 00032）至（GN-G。）。奖学金由荷兰科学研究组织"转化神经科学计划的研究生院"（022005019）至（CH）。 赠款从Prinses贝娅特丽克丝全宗（项目WAR08-12）和法语协会驳LES肌病为（PM-M。）;以及由荷兰科学研究组织的VENI奖学金（916.10.148）荷兰脑基金会的奖学金（FS2008（1）-28）和Prinses贝娅特丽克丝全宗（项目WAR08-12）（以ML ）。 我们感谢Jozien雅斯贝尔斯为她在B细胞通过流式细胞分选的帮助。

知情同意从研究的参与者获得的。这项研究是由伦理委员会。 1.分离外周血单个核细胞（PBMC的） <ol><li>离心机25毫升的参与者的肝素化血液，在900×g离心15分钟后，尽快在血液抽取之后。如果有血少，相应地缩减试剂。执行罩所有的下一步骤。 </li><li>血清转移到干净的管中。它可用于在后面的步骤为冷冻的PBMC或在自身免疫患者，以测试自身抗体。 </li><li>稀释血液用10ml的RPMI 1640培养基中，并通过上下抽吸悬浮细胞。 </li><li>添加包含多聚蔗糖和泛影酸钠（1.077克/ ml）的为15ml溶液到新的50ml管中。 </li><li>轻轻通过使的管紧密toget两个开口层上的血溶液的顶部在步骤1.4她直至两种液体来非常缓慢接触，然后滗出的PBMC悬浮液慢慢在50ml管中的顶部。此步骤是使PBMC中的良好分离是至关重要的。 </li><li>离心400 XG 20分钟步骤1.5获得不带刹车在RT管。 </li><li>离心后，回收用吸管出现在管中，其中包含的PBMC的中间部分的白圈。 </li><li>用25ml的RPMI 1640培养基洗细胞。 </li><li>离心300×g离心在RT 10分钟。 </li><li>弃去上清液，并用10ml的RPMI 1640离心机再次洗细胞如在步骤1.9。 </li><li>算上与纽鲍尔室的外周血单个核细胞如先前报道14。 </li><li>处理的PBMC作为在第2或将其存储以供以后使用如下:1000万的PBMC每离心管筒稀释在1ml的10％二甲基亚砜（DMSO）和90％的血清，在步骤1.2获得的结冰。冻结逐步长期STO愤怒在液氮中。 </li></ol> 2.染色的外周血单个核细胞CD22排序+和IgG +细胞经流式细胞仪<ol><li>板中的25cm 2细胞培养瓶中的PBMC用6ml完全RPMI 1640培养基中（补充有L-谷氨酰胺，10mM的HEPES缓冲液，50单位/毫升青霉素，50微克/ ml链霉素和胎牛血清的10％）的并让他们在37℃下回收的O / N的培养箱中在5％的CO 2。 </li><li>方框无菌FACS管，用无菌4％白蛋白的磷酸盐缓冲盐水（PBS）溶液O / N。用PBS洗两次的管中，用500微升的完全RPMI 1640培养基中的填充它们。使用这些管进行排序后恢复细胞。 </li><li>收集在管和离心机的PBMC，在400×g离心在RT 5分钟。 </li><li>悬浮细胞的标记缓冲液（参见步骤2.5和2.6），并指望他们14。的标记缓冲液是无菌的2％胎牛血清和P中1mM的乙二胺四乙酸（EDTA）BS。 </li><li>制备3荧光激活细胞分选（FACS）管与10 5个细胞每重悬它们在100微升标记缓冲液。这些管将成为定义排序的大门。 <ol><li>在第一个试管中加入5微升抗CD22抗体PerCP（建议在制造商的数据表），在第二管20微升抗IgG抗体的PE（建议在制造商的数据表），并没有在第三管。在30分钟期间在冰上孵育并在黑暗中。 </li></ol></li><li>悬浮5000000细胞在200微升标记缓冲液在FACS管中，加入10微升抗CD22 PerCP和40微升抗IgG抗体的PE。在30分钟期间在冰上孵育和暗细胞中。如果一些细胞更高的排序是期望的，扩大的所有试剂。 </li><li>离心细胞，在400×g离心以低速制动器为5分钟，在RT。 </li><li>轻轻倒出上清液。 </li><li>悬浮细胞在500μ升标记缓冲液。离心细胞，在400×g离心以低速制动器为5分钟，在RT。重复步骤2.7和2.8 </li><li>轻轻倒出上清和悬浮细胞在300微升RPMI完整的媒体。 </li><li>通过0.45微米的过滤器传递的细胞。细胞准备进行排序。 </li></ol> B细胞CD22 +和IgG 3排序+ <ol><li>使用3对照管来建立细胞的活力。在没有染色的控制，使用前向和大小散点图来定义淋巴细胞门控。与CD22 PerCP和抗IgG PE的控制有助于建立门，分配闸门。执行下列先前报道的数据15。 注:门是一组值的限制（边界）是用来隔离流式细胞仪事件的特定群体，在我们的细胞的情况下，从一个大集合。在一个分配门限制值的情况下，允许细胞计数的事件，细胞，这是内部的回收定义的边界:CD22 +和IgG +。 </li><li>作为在分类收集管中，使用封端管用500μl完全RPMI 1640培养基中，从步骤2.2获得的。 </li><li>进行排序双重利好15:CD22 +的IgG +。注意细胞在每种条件下的最终数量。板尽快排序在饲养细胞上的顶端细胞。 </li></ol> 4.辐照饲养细胞注:执行1之间的饲养细胞的制备 - 排序前3天。在96轮孔板每孔至少需要5000 WI38细胞。执行步骤4.1，在4.2引擎盖和4.4。 <ol><li>培养WI38细胞在37℃和5％的CO 2中的完全RPMI 1640培养基（相同的PBMC），直到单元的所需数目被达到。 </li><li>他们照射在50灰色，下面16之前描述的协议。一旦WI38细胞已被胰蛋白酶消化并重新悬浮在C进行照射omplete RPMI培养基，或者与WI38附着在培养瓶和照射后胰蛋白酶消化。按照方法通过胰蛋白酶细胞WI38的供应商表示。照射必要以覆盖所需的电镀的IgG + B细胞（总PBMC的1％，在步骤1.11计算）的孔的细胞数量。 </li><li>板含有5000 90微升照射在96轮孔板的每个孔WI38细胞。离开井板空的外排（因为它们蒸发更快），并且只使用60井板的中间电镀细胞。填补被诬陷板200微升PBS的外口井。 </li><li>将它们放置在一个培养箱中在37℃在5％的CO 2，直到需要。 </li></ol> 5.电镀排序外周血单个核细胞，EB病毒感染与成长<ol><li>淡化排序外周血单个核细胞，以板50细胞的50微升RPMI 1640完全培养基每口井在96轮孔板（以前机智镀ħWI38照射细胞）。请在装备EBV工作罩步骤。 注意:每孔50个细胞的感染被优化，并增加了似然产生的单克隆抗体8，但是，建议通过PCR来验证这如1,6-前所述。 </li><li>添加60微升EB病毒上清（含有3 - 4×10 8病毒拷贝/ ml）以每孔在96轮孔板17。注意应在EBV工作采取。 </li><li>加的CpG 1微克/毫升（ODN2006）每孔。 </li><li>离开细胞在培养箱中在37℃在5％的CO 2。 </li><li>直观地监视克隆一周后在显微镜下成长。 注意:B细胞生长的一些实例可以在结果部分下面可以看到。注意克隆的生长速率可能会发生变化，并且额外的时间可能需要开始看到一些细胞块中的孔的中间生长。 </li><li>第一届 2周后的Monitor克隆增长的光显微镜下进行第一次媒体交流会。慢慢吸管90微升培养基从井的顶部，并收集在一个干净的96孔板中。 （B细胞存在于井的底部，所以没有风险抽吸它们）。 <ol><li>加入每孔100μl的完全RPMI 1640培养基补充有CpG的（ODN2006）的1微克/毫升和IL2的50U /毫升。如果克隆的增长，通过分析这ELISA第一媒体交流在第6步。 </li></ol></li><li>监测克隆生长和3 次和采取90微升的媒体并加入100微升的完全RPMI 1640培养基补充有CpG的（ODN2006）的1微克/毫升和50 U / ml的再次生长的第 4周之后改变媒体IL2。使用回收的上清并监控IgG产生通过ELISA如在步骤6。 注:一个月后的克隆成长应该是很明显通过观察圆形细胞聚集塔吨通常在于圆底的中间孔。 </li><li>在此步骤中，以同样的方式改变媒体的前几周，但只用完全RPMI 1640培养基。一个很好的指标要知道正确的时刻来改变媒体是媒体的颜色，如果是淡黄色变成细胞需要一个媒体交换。 </li><li>当96-孔板含有大的克隆（约5×10 5个细胞），将其转移到一个24孔板的平坦井。添加300μl的完全RPMI 1640培养基中，以在新井。重悬96孔增长的克隆，移液器向上和向下几次，并转移到细胞的新井，均匀分布。在需要的时候添加新的媒体。 <ol><li>当在24孔板的孔中充满细胞（约5×10 6个细胞），转移至6孔板的平坦井。加入2ml完全RPMI 1640培养基中，以在新井。重悬的细胞在24孔板通过上下抽吸数次，然后分发给homogeneously在新井。 </li></ol></li><li>必要时添加的媒体。如果细胞不被维持在培养，沉淀细胞，在400×g离心5分钟，在RT。存储上清用于ELISA检测。用1×PBS中，离心再次在400×g离心洗细胞沉淀一次5分钟，并干燥保存于-80℃直至进行RNA提取。 <ol><li>当细胞在6孔板成为汇合（约20×10 6个细胞），转移到60毫米培养皿。加4 ml完全RPMI 1640媒体到新的板块。悬浮细胞在6孔板，转让为已完成的步骤是5.9和5.10。 </li></ol></li><li>在需要的时候添加新的媒体。在此步骤中，冻结细胞在10 - 90％的胎牛血清，30万元/ ml和10％的DMSO。如果较大的细胞数量都想要的，继续做大表面板扩容的克隆。 </li></ol> 6. ELISA方法的IgG抗体检测<ol><li>免除50微升/孔的ELISA板Ø˚F山羊的F（ab）2抗人类Fc抗体稀释1 200在包被缓冲液。包被缓冲液含有50毫的Na 2 CO 3的pH 9.6。 </li><li>密封板用塑料贴纸，并孵育1小时，在37℃。另外，在孵育4°CO / N。 </li><li>用200μl的洗涤缓冲液洗涤各孔6次。洗涤缓冲液含有0.05％Tween-20的1×PBS中。请勿触摸或划伤以及表面，该抗体与约束。 </li><li>用封闭缓冲液，100μl/孔阻塞。封闭缓冲液包含非脱脂奶粉在PBS中的4％。密封板并孵育1小时，在37℃。 </li><li>不洗。抛弃封闭液和巴掌板3次倒在纸巾上，除去残留的液体。 </li><li>孵育克隆的上清液和标准。 <ol><li>对于第一次测试，稀释步骤5.6或5.7 1/3 RPMI获得克隆上清。对于标准稀释用RPMI培养基人IgG的解决方案来获得:1000纳克/毫升，500纳克/毫升，250纳克/毫升，125纳克/毫升，62.5纳克/毫升，31.25毫微克/毫升，15.6纳克/毫升和空白。使用50微升/孔，37℃60分钟。分析克隆&#39;上清液来测试抗体亲和力的进一步的稀释，例如1/10或1/100。 </li></ol></li><li>作为洗净完成步骤6.3。 </li><li>使用的山羊F50μl/孔（AB）2抗人IgG Fc的过氧化物酶偶联的稀释1:20,000在培养缓冲液。孵育缓冲液含有在1×PBS中的1％BSA和0.02％吐温20。在37℃进行60分钟。 </li><li>作为洗净完成步骤6.3。 </li><li>加含有0.1％的3,3&#39;100μl/孔底物溶液，5,5&#39;-四甲基联苯胺（TMB）。孵育10分钟，直到稳定的蓝色形式在孔中。小心;不要等待太久！ </li><li>停止反应，加入50微升2 MH 2 SO 4。在反应停止后测量的吸收在450nm，在30分钟内。 注意:所有的克隆的upernatants超过坯料3个标准偏差将被认为是阳性对IgG的人抗体。为确认阳性克隆的该ELISA应至少重复三次，在同一克隆的不同上清。还丢弃非特异性交叉反应的可能性，建议，以确保没有信号时上清液孵育在未涂覆但受阻井。在该步骤中，检测分析以筛选感兴趣的抗体的抗原特异性可以进行到第7之前进行。 </li></ol>生产工艺中的IgG克隆的7 RNA分离和第一链cDNA合成<ol><li>只要生产的IgG通过ELISA证实，提取该克隆的RNA。 </li><li>从细胞步骤5.7或5.9日益增长的RNA提取，使用上标III细胞直接cDNA合成试剂盒，其允许获得的DNA从10,000个细胞一个细胞。 </li><li>从拉尔提取RNA细胞的数量蒙古包，或从存储在步骤5.11颗粒使用高纯度RNA分离试剂盒。 RNA提取的其他系统也可以是适合在此步骤。按照制造商的说明。 </li><li>为细胞数1×10 6和1×10 4利用反转录系统，按照制造商的指示。也可以用于其它系统。第一链cDNA合成。 </li></ol> 8. 1 和第 2 次 PCR为重的扩增和IgG抗体生成B细胞克隆轻链<ol><li>用细胞克隆，在步骤6，在步骤5.6和5.7和阳性所得的IgG抗体的ELISA中的cDNA，与表1中所列的引物进行PCR。 </li><li>设立独立的PCR每个IgG重，κ和λ链。使原液与正向和反向引物以相等的浓度。将它们添加到反应物在0.4μM的最终浓度。&lt;/ LI&gt; <li>使用1微升cDNA合成的进行1 ST的PCR。在这里，执行使用宝的Taq制造商的说明20微升反应。另外，使用其他聚合酶。 </li><li>放置1次的PCR以下循环条件下:94℃5分钟; （50个循环的:94℃30秒; 58℃，30秒，72℃45秒; 72℃，7分钟;让它在反应冷却，在4℃C.Include空白，检测到可能的污染。 </li><li>制备1×TBE（89毫摩尔Tris-硼酸和2mM EDTA）中。在100ml的1×TBE的体积添加1克琼脂糖（1％琼脂糖凝胶）的。加热在微波中约1分钟的溶液中，直到琼脂糖被溶解。再加入10mg / ml的溴化乙锭（或等价DNA染料）4微升混匀。 <ol><li>倒在托盘的内容，等到被聚合。运行5微升PCR混合物中的凝胶，以可视化的条带。重链片段也应该被大约400 - 550基点，而轻链应该在300 - 400基点。如果未检测到条带，同样进行到2 次 PCR。 </li></ol></li><li>使用1μL第一次PCR混合进行第2 次 PCR。使用，在步骤8.2，但使用的引物的合适的混合物（ 见表1）相同的试剂。为第2 次的PCR使用以下条件:94℃5分钟; （50个循环的:94℃30秒; 56.5℃30秒和72℃55秒）; 72℃7分钟;让它冷却下来，在4℃。包括在反应的空白，以检测可能PCR污染。 </li><li>准备琼脂糖凝胶在8.5描述。运行凝胶以可视化的PCR产物为8.5.1使用含有合适大小的片段克隆步骤9的扩增和产生抗体，在步骤10。 </li><li>序列的DNA，用10微升反应包含:100毫微克的第二 PCR，0。为2μM的引物或引物混合物，1μl的终止子和1微升缓冲液中的。执行测序反应在这些条件下:（25个循环:96℃，10秒; 50℃5秒;以及60℃下4分钟）; 60℃，7分钟;让它冷却下来，在4℃。 </li><li>净化用microspin G50柱按照生产商的说明，将测序反应。 18前述评估自动序列分析仪连接的顺序。电泳应清洁，对应于一个单一的免疫球蛋白序列。 注意:如果PCR产物显示不清或混合免疫球蛋白序列，请考虑使用TopoTA系统，以获得分离的序列，然后进行第9步克隆它们。 </li></ol> 9.克隆和IgG的产克隆的重和轻链的测序<ol><li>准备1微克载体DNA的pFUSE2ss-CLIg-HK，pFUSE2ss-CLIg-HL2和pFUSEss-CHIg-HG1。 </li><li>消化这些载体用合适的限制性酶。利用生态 RI和BSI为WI pFUSE2ss-CLIg-HK， 生态 RI和无毒二世pFUSE2ss-CLIg-HL2，与生态 RI和NHE我的pFUSEss-CHIg-HG1。使用3个单位的每个限制性酶的1微克载体DNA组成。 <ol><li>消化用一种酶并用PCR纯化试剂盒纯化消化产物。消化与第二酶和（根据制造商的协议）用凝胶提取试剂盒纯化。切的从琼脂糖凝胶感兴趣的片段。制备琼脂糖凝胶如在步骤8.5。 </li></ol></li><li>消化2 次 PCR产物的产生IgG的克隆: 生态 RI和BSI为WIκ链扩增， 生态 RI和无毒二世λ链放大，与生态 RI和NHE我的重链扩增。消化用一种酶和有P纯化消化产物CR纯化试剂盒。消化与第二酶和用PCR纯化试剂盒纯化消化产物。使用的酶和DNA量的单位相同，在步骤9.2。 </li><li>结扎PCR片段用T4 DNA连接酶16°CO / N以下制造商的建议向量的内部。用应为1:2的比例（载体:插入）。 </li><li>用1微升连接的改造DH5α细菌。按照DH5α制造商的条件进行改造。传播细菌在杀稻瘟素或zeocin的板，这取决于所使用载体的类型。孵育板O / N在37℃。 </li><li>长单菌落，并提取DNA用小量试剂盒，按照生产商的指示。使用单菌落生长，DNA的提取DNA小量试剂盒制造商的，建议。他们通过消化用于制备载体和插入的步骤9.2和9.3酶分析。 </li><li>序列的DNA，由全光照G百纳克载体为已完成的步骤8.8和8.9。 </li></ol> 10.生产抗体的HEK细胞<ol><li>生长在Dulbecco氏改良Eagle培养基（DMEM），补充有10％胎牛血清，1％L-葡萄糖，1％青霉素/链霉（DMEM +）以75％在150mm的细胞培养板中的汇合的HEK细胞。执行罩步骤10.1至10.7。 </li><li>转染前，交换介质以介质之前，但无胎牛血清。 </li><li>对于每一个重，轻链转染，制备转染混合物，用2.5毫升的DMEM（无血清），9微克与重链的载体，6微克与轻链的载体，以及100微升的聚乙烯亚胺（PEI ）解决方案（从1微克/微升股）。在室温下孵育15分钟。 </li><li>轻轻地加入2.5毫升10.3一步的HEK细胞在板准备转染混合物中。岩石板均匀分布。 </li><li>在cubate中的细胞培养箱中，在37℃，5％CO 2的24小时。 </li><li>无胎牛血清改变培养基介质。 </li><li>从板4天后收集培养基。 注意:在该介质中的抗体可用于表征它们的反应性在体外和体内 。 </li></ol>

<ol>
	<li>Vrolix, 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=Clonal+heterogeneity+of+thymic+B+cells+from+early-onset+myasthenia+gravis+patients+with+antibodies+against+the+acetylcholine+receptor.">Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.</a> J Autoimmun. 52, 101-112 (2014).</li><li>Yamashita, M., Katakura, Y., Shirahata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+generation+of+human+monoclonal+antibody.">Recent advances in the generation of human monoclonal antibody.</a> Cytotechnology. 55 (2-3), 55-60 (2007).</li><li>Pereira, K. M., Dellavance, A., Andrade, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+identification+of+autoantibodies+specific+to+systemic+autoimmune+rheumatic+diseases+in+high+throughput+operation:+Proposal+of+reliable+and+feasible+strategies.">The challenge of identification of autoantibodies specific to systemic autoimmune rheumatic diseases in high throughput operation: Proposal of reliable and feasible strategies.</a> Clin Chim Acta. 437, 403-410 (2014).</li><li>Losen, 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=Treatment+of+myasthenia+gravis+by+preventing+acetylcholine+receptor+modulation.">Treatment of myasthenia gravis by preventing acetylcholine receptor modulation.</a> Ann N Y Acad Sci. 1132, 174-179 (2008).</li><li>Smith, 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=Rapid+generation+of+fully+human+monoclonal+antibodies+specific+to+a+vaccinating+antigen.">Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.</a> Nat Protoc. 4 (3), 372-384 (2009).</li><li>Fraussen, J., 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+novel+method+for+making+human+monoclonal+antibodies.">A novel method for making human monoclonal antibodies.</a> J Autoimmun. 35 (2), 130-134 (2010).</li><li>Traggiai, E., 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=An+efficient+method+to+make+human+monoclonal+antibodies+from+memory+B+cells:+potent+neutralization+of+SARS+coronavirus.">An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.</a> Nat Med. 10 (8), 871-875 (2004).</li><li>Fraussen, J., 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=Autoantigen+induced+clonal+expansion+in+immortalized+B+cells+from+the+peripheral+blood+of+multiple+sclerosis+patients.">Autoantigen induced clonal expansion in immortalized B cells from the peripheral blood of multiple sclerosis patients.</a> J Neuroimmunol. 261 (1-2), 98-107 (2013).</li><li>Yamashita, 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=Different+individual+immune+responses+elicited+by+in+vitro+immunization.">Different individual immune responses elicited by in vitro immunization.</a> Cytotechnology. 40 (1-3), 161-165 (2002).</li><li>Hui-Yuen, J., Koganti, S., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cell+immortalization+for+monoclonal+antibody+production.">Human B cell immortalization for monoclonal antibody production.</a> Methods Mol Biol. 1131, 183-189 (2014).</li><li>Tiller, 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=Efficient+generation+of+monoclonal+antibodies+from+single+human+B+cells+by+single+cell+RT-PCR+and+expression+vector+cloning.">Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.</a> J Immunol Methods. 329 (1-2), 112-124 (2008).</li><li>Lanzavecchia, A., Corti, D., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monoclonal+antibodies+by+immortalization+of+memory+B+cells.">Human monoclonal antibodies by immortalization of memory B cells.</a> Curr Opin Biotechnol. 18 (6), 523-528 (2007).</li><li>Traggiai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+B+cells:+analysis+of+B+cell+repertoire+and+production+of+human+monoclonal+antibodies.">Immortalization of human B cells: analysis of B cell repertoire and production of human monoclonal antibodies.</a> Methods Mol Biol. 901, 161-170 (2012).</li><li>Strober, W., 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=Monitoring+cell+growth.+Curr+Protoc+Immunol+/+edited+by.">Monitoring cell growth. Curr Protoc Immunol / edited by.</a> John E. Coligan ... [et al.]. Appendix 3, Appendix 3A (2001).</li><li>Ibrahim, S. F., van den Engh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+cell+sorting.">Flow cytometry and cell sorting.</a> Adv Biochem Eng Biotechnol. 106, 19-39 (2007).</li><li>Leith, J. T., Padfield, G., Faulkner, L. E., Quinn, P., Michelson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+feeder+cells+on+the+X-ray+sensitivity+of+human+colon+cancer+cells.">Effects of feeder cells on the X-ray sensitivity of human colon cancer cells.</a> Radiother Oncol. 21 (1), 53-59 (1991).</li><li>Hui-Yuen, J., McAllister, S., Koganti, S., Hill, E., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Epstein-Barr+virus+growth-transformed+lymphoblastoid+cell+lines.">Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines.</a> J Vis Exp. (57), 3321 (2011).</li><li>Smith, L. 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=Fluorescence+detection+in+automated+DNA+sequence+analysis.">Fluorescence detection in automated DNA sequence analysis.</a> Nature. 321 (6071), 674-679 (1986).</li></ol>

作者宣称，他们有没有竞争的财务权益。

在这个手稿，所有步骤从人PBMC的IgG抗体的产生，详细介绍。该协议包括一些优势较以前的发布技术。的优点之一是，产生的抗体保持对应于原始对中的B细胞克隆的重链和轻链。 IgG抗体的鉴定可以在任何类型的人供体的工作要做，而且也没有必要对由于疫苗接种5免疫应答的恶化。使用成纤维细胞系WI38作为饲养细胞的，允许更快速检测的生长克隆的，因为它们在形态上不同，很容易区分，...

本文的目的是详细描述的方法来产生和表征从人外周血单核细胞（PBMC）中得到的人IgG单克隆抗体。 研究人类抗体的兴趣增加在研究许多不同的领域。特别是，许多研究小组有兴趣引起自身抗体1-3的病理。我们已经克隆和表征致病的自身抗体1。自身抗体的研究能够帮助确定他们的目标，并制定治疗策略， 例如 ，采用竞争性抗体4。另外，人抗体的研究也可以是在研究等领域， 即兴趣，以评估疫苗接种5后的免疫反应，以表征该被暴露，并成为抗特定病原体的6或研究个体的抗体分布曲线抗体是在自然剧目7,12。 几种技术已经开发了产生重组人单克隆抗体8-12;大多数的这些使用噬菌体展示和B细胞永生化。使用噬菌体展示已被广泛应用于新抗体13的发现。然而，它具有一个主要的缺点，即重链和轻链对人免疫球蛋白成为离解的过程中。生产的杂交瘤与人类B细胞或EBV转化的克服了这一缺点。 我们通过Toll样受体9（TLR-9）6,12使用胸腺B细胞的感染与EBV与多克隆B细胞刺激的组合。 在本文中，我们详细描述了我们使用的抗体人源抗体的发展，从PBMC隔离的所有步骤， 体外抗体生成一个完整的概述的技术。这个协议可用于任何类型的人IgG轮廓的分析。在我们的实验室中，B细胞产生IgG抗体已成功地从PBMC中的其余部分分选后分离。五十排序B细胞8然后可以镀在多孔板和由EB病毒和TLR-9的激活永久化，对于单一B细胞的克隆扩增。作为饲养细胞，从人胚胎肺组织的成纤维细胞已被使用，细胞系WI38，这有利于该永生化B细胞的可视化。从这些B细胞，免疫球蛋白的重和轻链的序列可以通过PCR来获得，并且该抗体"基因克隆中免疫球蛋白G的表达载体和体外生产。使用这种技术，单个的抗体与正好在供体中发现的相同的抗体序列可以研究。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>solution containing polysucrose and sodium diatrizoate</td>
		</tr>
		<tr>
			<td>FACSAria II cell sorter&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 U-bottom micro well plates&#160;</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Roswell Park Memorial Institute (RPMI) 1640 medium&#160;</td>
			<td>Gibco, Life Technologies</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>30% v/v EBV-containing supernatant of the B95-8 cell line&#160;&#160;</td>
			<td>ATCC</td>
			<td>CRL-1612</td>
			<td>3.4 x 108 copies/ml</td>
		</tr>
		<tr>
			<td>CpG2006&#160;</td>
			<td>Invivogen</td>
			<td>ODN 7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Wi38 cells&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>90020107</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin-2</td>
			<td>Roche&#160;</td>
			<td>10799068001</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates</td>
			<td>Greiner Bio-One, Microlon</td>
			<td>655092</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure F(ab&#39;)2 Fragment Goat Anti-Human IgG, Fc&#947; Fragment Specific (unconjugated)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-006-008</td>
			<td></td>
		</tr>
		<tr>
			<td>4% non-fat dry milk (Blotting Grade Blocker)&#160;</td>
			<td>Biorad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG&#160;</td>
			<td>Sigma</td>
			<td>I 2511</td>
			<td>HUMAN IgG purified Immunoglobulin, 5.6 mg/ml</td>
		</tr>
		<tr>
			<td>Goat F(ab)2 antihuman IgG Fc&#947; (conjugated with peroxidase (PO))</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-036-008</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA reader (Perkin Elmer 2030)&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td>2030-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5&#181;&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>309-035-095</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Cells Direct cDNA Synthesis System&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>18080-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems (ABI) GeneAm PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Pure RNA Isolation Kit&#160;</td>
			<td>Roche</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcription system kit&#160;</td>
			<td>Promega</td>
			<td>A3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Taq DNA Polymerase</td>
			<td>TAKARA</td>
			<td>R001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers (2&#956;l)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>Invitrogen</td>
			<td>15628-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantity One 4.5.2 (Gel Doc 2000)</td>
			<td>Biorad</td>
			<td>170-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>QIAGEN</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 cycle sequencing kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 ml reaction plate (MicroAMP Optical 96-well)</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetic analyser ABI300&#160;</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells (E. coli)</td>
			<td>Invitrogen</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG1 (4493 bp)</td>
			<td>Invivogen</td>
			<td>pfusess-hchg1</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG4 (4484 bp)&#160;</td>
			<td>Invivogen</td>
			<td>pfusess-hchg4</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hk (3875 bp)</td>
			<td>Invivogen</td>
			<td>pfuse2ss-hclk</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hl2 (3883 bp)&#160;</td>
			<td>Invivogen</td>
			<td>pfuse2ss-hcll2</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Invitrogen</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar)</td>
			<td>Invivogen</td>
			<td>fas-zn-s</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB)</td>
			<td>Invivogen</td>
			<td>fas-zn-l</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Miniprep kit&#160;</td>
			<td>Omega Bio Technology</td>
			<td>D6942-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop (ND1000 Spectrophotometer)</td>
			<td>Nanodrop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar)</td>
			<td>Invivogen</td>
			<td>fas-bl-s</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB)</td>
			<td>Invivogen</td>
			<td>fas-bl-l</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101S</td>
			<td>20,000 U/ml, in 10x NEBuffer EcoRI</td>
		</tr>
		<tr>
			<td>NheI</td>
			<td>New England Biolabs</td>
			<td>R0131S</td>
			<td>10,000 U/ml, in 10x NEBuffer 2.1</td>
		</tr>
		<tr>
			<td>2-Log DNA ladder</td>
			<td>New England Biolabs</td>
			<td>N3200S</td>
			<td>0.1-10.0 kb, 1,000 &#956;g/ml</td>
		</tr>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180S</td>
			<td>10,000 U/ml, in 10x CutSmart Buffer&#160;</td>
		</tr>
		<tr>
			<td>BsiWI</td>
			<td>New England Biolabs</td>
			<td>R0553S</td>
			<td>10,000 U/ml, in 10x NEBuffer 3.1</td>
		</tr>
		<tr>
			<td>AvrII</td>
			<td>New England Biolabs</td>
			<td>R0174S</td>
			<td>5,000 U/ml, in 10x CutSmart Buffer&#160;</td>
		</tr>
		<tr>
			<td>FastAP Thermosensitive Alkaline Phosphatase</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>1 U/&#181;L, in 10x FastAP Buffer</td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells&#160;</td>
			<td>Invitrogen</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human IgG</td>
			<td>BD Pharmingen</td>
			<td>555787</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD22, PerCP-Cy5.5, Clone: HIB22</td>
			<td>Fisher scientific</td>
			<td>BDB563942</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit&#160;</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

染色CD22和IgG阳性细胞之后的分选门控示于图1在该图像中的双阳性细胞的面积。 -产生IgG抗体的B细胞-被选择为所有这些细胞在一个单独的试管中进行排序。在分析中，总的PBMC约1％的对应于这种双重阳性群体。得到的分选的细胞的数目将取决于在第1获得的细胞的数量。 5周EB病毒永生化和CpG（ODN2006）刺激后的不同结果示于图2中的生长克隆的检测容易?...

Watch this Scientific Journal Video about Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells at JoVE.com

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

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-recombinant-human-igg-monoclonal-antibodies-from

Research

JoVE Journal

Immunology and Infection

18.3K 次观看.  Maastricht University. 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.

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...