我们感谢细胞仪设备 "CytoCell" (SFR Santé, Biogenouest, 南特) 的专家技术援助。我们还感谢所有的重组蛋白生产 (P2R) 和冲击平台 (INSERM 1232, SFR Santé, Biogenouest, 南特) 的工作人员的技术支持。我们感谢伊曼纽尔 Scotet 和理查德 Breathnach 对手稿的建设性评论。这项工作的财政支持 IHU-Cesti 项目资助的« Investissements 艾文莉»法国政府计划, 由法国国家研究机构 (ANR-10-IBHU-005) 管理。IHU-Cesti 项目也得到了南特 Métropole 和 Région 支付 de la 卢瓦尔的支持。这项工作是在国家研究机构通过对未来项目 ANR-11-LABX-0016-01 的投资支持的 LabEX 总检办方案的背景下实现的。

根据当地道德委员会 (景区法国, EFS, 南特, 程序采样 NTS-2016-08), 所有的人外周血样本都是在知情同意后从匿名成年献血者那里获得的。
1. 人外周血单个核细胞的分离
注: 起始物料可为总人外周血或 cytapheresis 样。样品不应超过8小时, 并辅以抗凝血剂 (例如, 肝素)。
<ol><li>稀释血液与2容量 (人的外周血) 或5容量 (cytapheresis 样品) RPMI (罗斯威尔公园纪念学院) 媒介。</li>
	<li>在50毫升锥形管中仔细地将35毫升稀释的细胞悬浮液放在15毫升的密度梯度介质上。使尽可能多的管, 以分配所有稀释的血液。离心机在 1290 x g 为25分钟的室温在一个摆动斗转子与刹车。</li>
	<li>在密度梯度介质和等离子体层之间的界面上仔细地吸取单个核细胞层, 并将细胞转移到新的50毫升锥形管上。</li>
	<li>用 RPMI 介质填充管, 在室温下在 1290 x g 处混合和离心10分钟。小心地除去上清液。</li>
	<li>重20毫升 RPMI 培养基中的细胞颗粒, 离心 200 x g, 10 分钟取出血小板。小心地除去上清液。重复此步骤一次。</li>
	<li>用10% 胎牛血清 (FBS) 重 RPMI 培养基中的细胞颗粒。 注: 直接进行聚丙烯标记或将细胞保持在4° c 过夜, 浓度为 107细胞/毫升。</li>
</ol>2. 聚丙烯与银特异 B 细胞的磁性富集
<ol><li>在1:4 至化抗原单体的摩尔比下加入 PE 或 APC 标记的优质亲和或 BV421-labeled 亲和, 制备银体。在三单独的部分中加入适量的亲和共轭, 在室温下每10分钟加一分。</li>
	<li>在15毫升锥形管中分布单元 (最多 3 x 108 ), 在460分钟内将离心机放在每根 x g 处。 注意: 下面的协议是一个管。</li>
	<li>重200µL (磷酸缓冲盐水) 与 2% FBS 的细胞颗粒。添加 PE, APC 和 BV421-conjugated 体, 每一个最终浓度为10µg/毫升。拌匀, 室温孵育30分钟。</li>
	<li>用 PBS 制备 0.5% BSA (牛血清白蛋白) 和 EDTA (乙酸酸) 2 毫米的冰冷细胞分离缓冲液 (SB)。</li>
	<li>用离心法在 460 x g 处增加10毫升的颗粒细胞, 5 分钟。</li>
	<li>重细胞在500µL 冰冷 SB 中添加50µL 抗 PE 和50µL 的反 APC 磁性微。</li>
	<li>拌匀, 孵育20分钟, 在4° c。</li>
	<li>重复步骤2.5 次。</li>
	<li>清除上清液仔细 andresuspend 细胞颗粒在 2 x 108细胞/毫升的浓度在冰寒 SB。</li>
	<li>平衡在磁铁上放置一个大的磁性柱子, 有3毫升的。</li>
	<li>将电池悬挂从2.9 转移到平衡柱顶部, 使其完全耗尽。关键步骤: 为了避免柱状结块, 通过70µm 尼龙网格。</li>
	<li>将含有3毫升冰冷的细胞的管子冲洗干净, 将缓冲器直接转移到柱子上 (如过滤, 冲洗70µm 尼龙网)。</li>
	<li>当缓冲区已完全排入该列时, 将另一3毫升冰寒的人加到柱子上。</li>
	<li>重复步骤 2.13, 再进行3毫升洗涤。</li>
	<li>从磁铁中取出柱, 并在15毫升锥形收集管上放置。</li>
	<li>把5毫升冰冷的 SB 放在柱子顶部, 然后用柱塞将电池从柱上冲出。重复此步骤一次。</li>
	<li>离心收集的细胞 (丰富相关的聚丙烯阳性细胞分数) 在 460 x g 5 分钟, 并进行额外的抗体染色。 注: 池收集的细胞从所有管, 如果合适的。</li>
</ol>3. Ag 特定人 B 细胞的染色和细胞分类
<ol><li>重的细胞颗粒与抗人抗体鸡尾酒对 CD3 (最后稀释: 1:20), CD19 (1:20), CD14 (1:50), CD16 (1:50), 和 7AAD (1:1000) 在最后的容量100µL PBS 与 2% FBS。孵育30分钟, 在4° c。</li>
	<li>在细胞中加入10毫升的 PBS, 在 460 x g 处离心5分钟, 消除上清液。重复此步骤一次。重200µL PBS 中的细胞颗粒, 过滤到70µm 尼龙网。</li>
	<li>在单元格浏览细胞仪上继续对100µm 喷嘴和 20 psi 压力不超过1000事件/秒9的单个单元格级别上的特定 B 单元格进行排序。 注意: 使用的浇口策略显示在图 1.单元格首先在 CD14-CD16-7AAD 单元格中进行封闭, 以排除单核细胞、NK 和死单元 (未在 图 1中显示)。然后, 通过 PE 和 APC 相关的 Ag-体, 在双染色细胞上进行门控之前, 选择了 CD19+ B 细胞。非特异性 B 细胞被排除在 BV421 阴性细胞 (不由不相关的 Ag-聚丙烯) 的门。</li>
	<li>将单个 B 细胞收集到8条 PCR 管中, 之前填充有10µL 1x PBS 和10单位的核糖核酸抑制剂, 放在96管内的架子上。立即将 PCR 管冷冻在-80 ° c。 注: 在细胞仪仪器上选择的分类掩模为: 0、纯度面膜:32、相掩膜: 0. 如果需要, 这种单细胞沉积物可以调整为 96-或 384-好的 PCR 板。单 B 细胞必须尽快冷冻, 如果需要, 可以在-80 ° c 下留几个星期。</li>
</ol>4. 单细胞 rt-pcr
<ol><li>溶解细胞通过直接加热冷冻样品在70° c 的 PCR 带在干燥浴5分钟。</li>
	<li>传输带到冰和进行反向转录 (RT) 通过增加10µL 每管2x 主混合缓冲区包含1毫米 dNTP, 25 µg/毫升 oligod (T) 底漆, 5 µM 随机 hexamers, 20 单位核糖核酸抑制剂, 和400单位的反转录酶。 注意: 细胞解冻后, 必须迅速进行 RT, 以避免 RNA 降解。</li>
	<li>孵育在25° c 为5分钟, 允许随机 hexamers 到杂交, 然后孵育1小时在50° c, 并完成与孵化在95° c 为3分钟停止反应。</li>
	<li>为可变重 (vH) 和卡伯 (vLκ) 或λ光链 (vLλ) 的每个区域编码进行两轮嵌套 PCR 扩增。 注: 另外, RT 样品可在-20 ° c 冷冻并保存一周。<ol>
<li>在第一轮 PCR, 增加3µL 的 cDNA 为40µL 最后的体积, 包含1.5 毫米氯化镁2, 0.25 毫米 dNTPs, 2.5 单位的 DNA 聚合酶, 和 200 nM 的外部底漆 (见图 2和材料表的入门序列)。 注: 外底漆组合物的组成。对于重链放大: 4 前引物 (5 ' 肥厚的混合) 和2反向底漆 (3 ' CµCγ混合)。为轻卡伯链放大, 3 前引物 (5 ' LV | 混合) 和1反向底漆 (3 ' Cκ543-566)。对于光λ链放大, 7 前引物 (5 ' LVl 混合) 和1反向底漆 (3 ' Cl)。 注: 引物是由分蘖et al.设计的, 用于放大所有重和轻链家族基因5。</li>
		<li>应用以下循环条件:94 ° c 为 4 min, 跟随40周期三十年代在94° c, 三十年代在58° c 为 VH 和 VLκ (60 ° c 为 VLλ), 和五十五年代在72° c, 以最后的伸长步在72° c 为 7 min。</li>
		<li>对于第二轮 PCR, 使用3µL 的第一个放大产品的最后体积40µL 含有1.5 毫米氯化镁2, 0.25 毫米 dNTPs, 2.5 单位的 DNA 聚合酶, 和 200 nM 内引物含有限制酶点的克隆成表达向量 (请参见图 2和材料表中的入门序列)。 注: 内底漆组合物。对于重链放大: 9 前引物 (5 ' AgeIVH 混合) 和3反向底漆 (3 ' SalIJH 混合)。对于轻卡伯链放大, 9 前引物 (5 ' AgeIV | 混合) 和3反向底漆 (3 ' BsiWIJκ混合)。对于光λ链放大, 6 前引物 (5 ' AgeIVl 混合) 和1反向底漆 (3 ' XhoICl)。 注: 引物是由分蘖et al.设计的, 用于放大所有的重和轻链家族基因5。</li>
		<li>应用以下循环条件:94 ° c 为 4 min, 跟随40周期三十年代在 94, 三十年代在58° c 为 VH 和 LCκ (60 ° c 为 LCλ) 和四十五年代在72° c 以最后的伸长步在72° c 为 7 min。</li>
	</ol>
</li>
	<li>通过在1.5% 琼脂糖凝胶上迁移5μ l vH、vLκ和 vLλ的 PCR 产物 (500 bp 产品用于可变重链编码段和 350 bp 产品用于可变光链编码段), 确定正井。</li>
	<li>利用超滤膜提纯 pcr 产物, 纯化剩余的35µL pcr 产物。</li>
	<li>洗 PCR 产品与60µL 的 H2O。</li>
</ol>5. 表达式克隆
<ol><li>
准备插入 注: 在含有适当限制酶缓冲剂的100µL 的最后一卷中, 对每个纯化的 PCR 产物进行消化60µL。<ol>
<li>对于 vH PCR 产品, 增加50单位的 AgeI 和50单位的贝里沙, 并孵化1小时37° c。</li>
		<li>对于 vLλ PCR 产品, 增加50单位的 AgeI 和50单位的 XhoI, 并孵化1小时37° c。</li>
		<li>对于 vLκ PCR 产品, 增加50单位的 AgeI 和孵化1小时在37° c。用 96 PCR 膜和洗60µL 的µL 洗在µL 限制酶缓冲的 100 BsiWI 的最后一卷中, 消化了 60 BsiWI, 并在50° c 下孵育 1 h.</li>
		<li>在80° c 加热15分钟, 使酶钝化。</li>
	</ol>
</li>
	<li>
表达载体的制备 注: vH PCR 产物在一个表达载体 (HCγ1) 被克隆包含恒定的重链 Cγ1 IgG1。vLκ PCR 产物在含有恒光链 Cκ的表达载体 (LCκ) 中被克隆。vLλ PCR 产物在含有恒光链 Cλ的表达载体 (LCλ) 中被克隆。<ol>
<li>执行以下消解:<ol>
<li>混合1µg 的表达载体 HCγ1与10单位的 AgeI 和10单位的贝里沙酶的总容量20µL 的限制缓冲区的制造商建议, 并孵化 1 h 在37° c。</li>
			<li>混合1µg 的表达载体 LCκ与10单位的 AgeI 限制酶在总容量20µL 的限制缓冲区的制造商建议, 并孵化 1 h 在37° c。添加10单位的 BsiWI 限制酶, 并孵育1小时在55° c。</li>
			<li>混合1µg 的表达载体 LCλ与10单位的 AgeI 和10单位的 XhoI 酶的总容量20µL 的限制缓冲区的制造商建议, 并孵化 1 h 在37° c</li>
		</ol>
</li>
		<li>将每个消化混合物在65° c 下加热10分钟, 使限制酶钝化。</li>
		<li>用2µL (100 ng) 的相应线性化表达载体, 在20µL 1x dna 连接缓冲器的总容积中, 在1° c 的夜间孵育, 用4单位 T4 dna 连接, 进行结扎5µL 的 PCR 产物。 注: 另外, 结扎是在4° c 下进行3小时。</li>
	</ol>
</li>
	<li>
克隆
	<ol>
<li>Electroporate 1 µL 20 µL 结扎合剂成45µL 自制 electrocompetent TOP10大肠杆菌(目前的分子生物学协议, 1.8. 4-1. 8.8)。立即在37° c 的水浴中加入500µL 2X 的培养基, 孵育30分钟。在37° c 的夜间孵育。</li>
		<li>对于每一个转换, 筛选八殖民地的 PCR。<ol>
<li>在冰上设置反应预混料如下:45 µL 5x PCR 缓冲, 12 µL 氯化镁2 (25 毫米), 4 µL dNTPs (从每股包含10毫米), 10 µL 前引物 (股票10µM) 杂交到向量序列 (底漆 Ab-vec-感觉)和10µL 的反向底漆 (股票10µM) 的目标是恒定的重或轻链区域 (参见材料表的底漆序列)。用 H2O 组成200µL 的最终卷。然后加入4µL 的 DNA 聚合酶 (5 U/µL)。</li>
			<li>在冰上分布25µL 反应预混料每8条带 PCR 管。</li>
			<li>用一根无菌牙签来提取单个的菌落。将牙签贴在2x 的 TY 氨苄西林板上, 形成一个复制板, 然后将其浸入 PCR 反应管中。</li>
			<li>在以下循环条件下执行筛选 PCR:94 ° c 为 10 min, 跟随25周期三十年代在94° c, 三十年代在55° c 和五十年代在72° c。</li>
			<li>通过在1.5% 琼脂糖凝胶上迁移5µL 的 PCR 产物来鉴别阳性菌。 注意: 阳性菌落为重链向量和 600 bp 为轻链向量提供了一个 PCR 产品大约 850 bp。</li>
		</ol>
</li>
		<li>接种四阳性菌落从复制板到2毫升的 LB 培养基和孵化过夜在37° c 与晃动 200 rpm。</li>
		<li>使用质粒纯化试剂盒从液体培养基中提取粒, 如制造商所示。</li>
		<li>通过 vec 感引物的桑格测序, 验证变量域的正确插入。 注: 质粒与正确的插入 (编码 HC 和相应的 LC) 是 cotransfected 到293A 细胞的分泌。用 ELISA 法测定小规模生产抗体的特异性。然后, 在适用的情况下进行大规模生产。</li>
	</ol>
</li>
</ol>6. 抗体的生产
<ol><li>
小规模生产专用检测
	<ol>
<li>在转染前的前一天, 种子1.5万人胚胎肾脏 293A (HEK 293A) 细胞在200µL Dulbecco 氏改良鹰培养基 (DMEM) 补充 10% FBS 每井 96-井板。在 co2孵化器 (5% co2) 中, 在37° c 的夜间孵育。</li>
		<li>用线性亚衍生物作为转染试剂, 在含有 10% FBS 的 DMEM 培养基中 Cotransfect 293A 细胞。 注: 在 triplicates 中进行转染。下面的协议是一个平底96井板的一个井。<ol>
<li>将0.5 µL 的 DNA 转染试剂稀释为150毫米氯化钠10µL。稀释0.125 µg 的 vH 和0.125 µg 表达载体为10µL 150 毫米氯化钠。漩涡每稀释十年代。</li>
			<li>在10µL dna 溶液中加入10µL 稀释的 dna 转染试剂。十五年代涡旋, 室温孵育15分钟。加入20µL 混合滴明智的293A 细胞, 并通过轻轻旋转的板块混合。</li>
		</ol>
</li>
		<li>在无血清培养基中, 在转染和培养细胞5天后更换培养基16小时。 注: 使用无血清的培养基, 以避免血清中的抗体污染的产生。</li>
		<li>通过离心 460 x g 去除细胞和碎片5分钟。</li>
		<li>收获清通过吸入多通道吸管和转移到一个 V 底96井板。</li>
		<li>在4° c 的情况下, 每晚在100µL, 重组 elisa/ELISPOT 涂层缓冲 1x, 在 96-井 elisa 版中最终浓度为2µg/毫升。请参见9中的示例。</li>
		<li>10% FBS DMEM 介质2小时37° c 的块井</li>
		<li>添加50µL 清转染的293A 细胞从步骤 6.1.5, 并孵化2小时 RT。</li>
		<li>添加100µL 的抗人 IgG Ab 共轭的辣根过氧化物酶 (HRP) 酵素在1µg/毫升和孵化1小时室温。添加50µL 显色基底 (TMB), 孵育20分钟。</li>
		<li>在分光光度计上读取 450 nm 的光学密度。 注: ELISA 法显示的特异抗体可在大范围内进一步制备, 并在蛋白 A 柱上纯化, 对人类 IgG 具有亲和力。</li>
	</ol>
</li>
	<li>
特抗体的规模化生产与提纯
	<ol>
<li>在转染前的前一天, 种子 6 x 106人胚肾 293A (HEK 293A) 细胞进入175厘米2烧瓶, 含25毫升 DMEM 补充 10% FBS。在 co2孵化器 (5% co2) 中, 在37° c 的夜间孵育。 注: 转染时细胞应为70% 汇合。</li>
		<li>用线性亚衍生物作为转染试剂, 在含有 10% FBS 的 DMEM 培养基中 Cotransfect 293A 细胞。<ol>
<li>将50µL 的 DNA 转染试剂稀释为150毫米氯化钠450µL。稀释10µg 的 vH 和10µg 表达载体为500µL 150 毫米氯化钠。漩涡十年代每稀释。</li>
			<li>在 dna 溶液中加入稀释的 dna 转染试剂。十五年代涡旋, 室温孵育15分钟。加入1毫升混合滴明智的细胞, 并通过轻轻旋转的烧瓶混合。</li>
		</ol>
</li>
		<li>在无血清培养基中, 在转染和培养细胞5天后更换培养基16小时。</li>
		<li>用25毫升吸管抽吸培养基。</li>
		<li>通过离心 460 x g 去除细胞和碎片5分钟。</li>
		<li>在4° c 的快速蛋白液相色谱 (FPLC) 系统中, 用1毫升的蛋白 a 涂上一层蛋白质纯化抗体。<ol>
<li>平衡蛋白质 A 琼脂专栏与20毫米磷酸盐缓冲 (pH 值 7)。</li>
			<li>使用1.22 µm 过滤器从6.2.5 过滤细胞培养基, 然后是0.45 µm 过滤器。</li>
			<li>将筛选的介质加载到列上。</li>
			<li>用20毫米磷酸盐缓冲液 (ph 7) 和洗0.1 米的柠檬酸盐缓冲液 (ph 3.5) 洗涤。</li>
			<li>在吸收分光光度计上读取 280 nm 的光学密度, 并立即中和含有 1 M 三缓冲器 (pH 9) 的含蛋白质分数。</li>
		</ol>
</li>
		<li>透析纯化 Ab 在4° c 一夜的磁带与3.5K 分子量的防 PBS 缓冲 (pH 7.4)。</li>
		<li>由0.2 µm 过滤和控制纯度的大小-排除色谱, 如制造商所示。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Buss, N. A., Henderson, S. J., McFarlane, M., Shenton, J. M., de Haan, L. <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+therapeutics:+history+and+future.">Monoclonal antibody therapeutics: history and future.</a> Curr Opin Pharmacol. 12 (5), 615-622 (2012).</li><li>Park, J., Kwon, M., Shin, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+inhibitors+for+cancer+treatment.">Immune checkpoint inhibitors for cancer treatment.</a> Arch Pharm Res. 39 (11), 1577-1587 (2016).</li><li>Pendley, C., Schantz, A., Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+therapeutic+monoclonal+antibodies.">Immunogenicity of therapeutic monoclonal antibodies.</a> Curr Opin Mol Ther. 5 (2), 172-179 (2003).</li><li>Kwakkenbos, M. 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=Generation+of+stable+monoclonal+antibody-producing+B+cell+receptor-positive+human+memory+B+cells+by+genetic+programming.">Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.</a> Nat Med. 16 (1), 123-128 (2010).</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>Franz, B., May, K. F., Dranoff, G., Wucherpfennig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+and+isolation+of+rare+memory+B+cells+with+antigen+tetramers.">Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.</a> Blood. 118 (2), 348-357 (2011).</li><li>Morris, L., 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=Isolation+of+a+human+anti-HIV+gp41+membrane+proximal+region+neutralizing+antibody+by+antigen-specific+single+B+cell+sorting.">Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting.</a> PLoS One. 6 (9), e23532 (2011).</li><li>Iizuka, 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=Identification+of+cytomegalovirus+(CMV)pp65+antigen-specific+human+monoclonal+antibodies+using+single+B+cell-based+antibody+gene+cloning+from+melanoma+patients.">Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients.</a> Immunol Lett. 135 (1-2), 64-73 (2011).</li><li>Ouisse, L. H., 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=Antigen-specific+single+B+cell+sorting+and+expression-cloning+from+immunoglobulin+humanized+rats:+a+rapid+and+versatile+method+for+the+generation+of+high+affinity+and+discriminative+human+monoclonal+antibodies.">Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.</a> BMC Biotechnol. 17 (1), 3 (2017).</li><li>Kerkman, P. F., 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=Identification+and+characterisation+of+citrullinated+antigen-specific+B+cells+in+peripheral+blood+of+patients+with+rheumatoid+arthritis.">Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis.</a> Ann Rheum Dis. , (2015).</li><li>Hamilton, J. 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=General+Approach+for+Tetramer-Based+Identification+of+Autoantigen-Reactive+B+Cells:+Characterization+of+La-+and+snRNP-Reactive+B+Cells+in+Autoimmune+BXD2+Mice.">General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.</a> J Immunol. 194 (10), 5022-5034 (2015).</li><li>Sanjuan Nandin, I., 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=Novel+in+vitro+booster+vaccination+to+rapidly+generate+antigen-specific+human+monoclonal+antibodies.">Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies.</a> J Exp Med. , (2017).</li><li>Bowers, P. 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=Mammalian+cell+display+for+the+discovery+and+optimization+of+antibody+therapeutics.">Mammalian cell display for the discovery and optimization of antibody therapeutics.</a> Methods. 65 (1), 44-56 (2014).</li></ol>

作者没有什么可透露的。

所提出的协议是一种强有力的方法, 产生的人抗体直接从 Ag 特定的 B 细胞循环血液。它结合了三重要的方面: (i) 使用聚丙烯相关的磁性富集, 它允许对甚至稀有的银结合 B 细胞的ex 体内隔离;(二) 采用三 ag 体 (两个相关的和一个不相关的) 的门控策略, 用三种不同的荧光来隔离, 用流式细胞仪, 只对所需的 ag 表达一个 BCR 特异的 B 细胞;(iii) 用 rt-pcr 技术在单细胞水平上重建相应的重组单克隆人?...

An erratum was issued for: <a href="https://www.jove.com/video/56508/generation-discriminative-human-monoclonal-antibodies-from-rare">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood</a>.
The Affiliations section was corrected from:
Marie-Claire Devilder1,2,&#160;M&#233;linda Moyon1,2,&#160;Xavier Saulquin1, Laetitia Gautreau-Rolland1 
1Centre R&#233;gional de Recherche en Canc&#233;rologie et Immunologie Nantes/Angers, INSERM 1232, LabEx IGO, Universit&#233; de Nantes 
2CHU Nantes
to:
Marie-Claire Devilder1,2,3,&#160;M&#233;linda Moyon1,2,3,&#160;Xavier Saulquin1,2, Laetitia Gautreau-Rolland1,2&#160; 
1CRCINA, Inserm, CNRS, Universit&#233; d&#39;Angers, Universit&#233; de Nantes 
2LabEx IGO, &#34;Immunotherapy, Graft, Oncology&#34; 
3CHU, Nantes, France

An erratum was issued for: <a href="https://www.jove.com/video/56508/generation-discriminative-human-monoclonal-antibodies-from-rare">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood</a>

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

这里描述的方法允许快速和多才多艺的生产完全人的单克隆抗体 (抗体) 反对期望抗原 (Ag)。抗体是许多基础研究应用的重要工具在体外和在体内: 流式细胞术, 组织学, 西部印迹, 和阻断实验, 例如。此外, 抗体正在越来越多地用于治疗自身免疫性疾病, 癌症, 并控制移植排斥1。例如, anti-CTLA-4 和 anti-PD-1 (或 anti-PD-L1) 抗体最近被用作癌症治疗的免疫检查站抑制剂2。
第一抗体是由免疫球蛋白 (Ig) 分泌的杂交从脾细胞中获得的小鼠或大鼠。然而, 强的免疫反应对鼠或大鼠抗体阻碍他们的治疗用途在人, 由于他们的快速的清除和可能的感应性过敏反应3。为了解决这个问题, 动物蛋白序列的抗体已部分被人类的替代, 以产生所谓的嵌合体的小鼠-人类或人性化的抗体。然而, 这一战略只部分降低了免疫原性, 同时大大增加了成本和生产的时间尺度。一个更好的解决方案是直接从人类 B 细胞中生成人抗体, 并提供了几种策略。其中之一是使用噬菌体或酵母显示。这包括在噬菌体或酵母中的随机人类 Ig 重和轻链的组合库中显示可变域, 并使用特定的兴趣抗原进行选择步骤。这一战略的一个主要缺点是, 重和轻链随机关联, 导致一个非常大的增加, 产生抗体的多样性。获得的抗体不太可能与天然免疫应答对某一特定的 Ag 产生的反应相对应。此外, 人类蛋白质折叠和后转化的修改没有系统地复制在原, 甚至在酵母。第二个人类单克隆生物的生产方法是永生的自然人 B 细胞, 由 eb 病毒感染或表达的抗凋亡因素 BCL-6 和总格-XL4。然而, 这种方法仅适用于记忆 b 细胞, 而且效率低下, 需要筛选出大量的单克隆抗体生成永生化 b 细胞, 以确定少数 (如果有的话) 克隆人的抗原特异性。因此, 这种方法既费钱又耗时。
一个新的协议最近被描述用于生产人类抗体从孤立的单一 B 细胞5。它依赖于一个优化的单细胞逆转录聚合酶链反应 (RT PCR) 放大的重和轻链编码段从一个单一的排序 B 细胞。其次是在一个真核表达系统中, 这些片段的克隆和表达, 从而允许重建一个完全人类的单克隆。该协议已成功地用于从接种疫苗的捐献者 B 细胞开始。在接种疫苗数周后, 细胞获得了针对所需 Ag 的 B 细胞的更高频率, 从而限制了筛查6所需的时间。其他完全人类抗体也已经产生了艾滋病毒的+ (人体免疫缺陷病毒) 感染患者的7和黑色素瘤患者8。尽管有这些进展, 仍然没有可用的程序, 可以独立于其记忆表型或频率的 Ag 特定 B 细胞的分离。
这里描述的过程导致了有效的体外分离人循环 B 细胞基于他们的 BCR 特异性, 其次生产完全人抗原特异抗体在高产和以低筛选时间. 该方法不限于记忆 b 细胞或抗体分泌 b 细胞后诱导免疫应答, 但也可以应用于人类的幼稚 b 细胞的剧目。它运作甚而从 Ag 特定 B 细胞存在在非常低频率是它的效率的一个好征兆。该方法的原理如下: 外周血单个核细胞 (PBMC) 染色的两个体呈现的抗原的兴趣, 每个标记有不同的荧光 (例如, 藻 (PE) 和复方 (APC)),和第三聚丙烯提出一个紧密相关的抗原共轭与第三荧光 (如, 高明紫 421 (BV421))。为了丰富抗原结合细胞, 细胞然后孵化的珠子涂敷反 PE 和反 APC Abs, 并排序在细胞分离列。选择 PE+ APC+单元格部分, 用各种不同的抗体特定的 PBMC 细胞类型进行染色, 以允许识别 B 细胞, 并进行流式细胞仪细胞分类。B 单元格是 PE+和 APC+, 但明亮的紫罗兰-是孤立的。此步骤计数器-选择不是 B 细胞或不绑定到 tetramerized 抗原的细胞, 但要绑定到 pe 或 apc (这些单元格将是 pe+ apc-或 pe apc+), 或者是使用体的非抗原部分 (这些单元格将 BV421+)。B 细胞不是高度具体的表位的兴趣也是计数器选择在此步骤 (这些单元也将是 BV421 的+)。因此, 这种方法可以纯化高特异 b 细胞表达 b 细胞受体 (BCRs) 能够区分两个非常密切的相关抗原。在试管中收集单个特定的 B 细胞, 并将其 PCR 扩增的 Ig 基因 (补充脱氧核糖核酸) 克隆出来, 由人类细胞系作为分泌的 IgG 抗体表达。
作为概念的证明, 本研究描述了人类抗体的有效生成, 它能识别出由一个主要的组织相容性复合 i 类 (MHC i) 分子所呈现的肽, 并能区别这一肽和其他加载在同一MHC I 等位基因。虽然这个 ag 的复杂程度是重要的, 这个方法允许 (i) ag 特定抗体的高收率的补救;(二) 生产抗体能够区分两个结构密切 Ags。这种方法可以扩大到接种疫苗或感染的病人没有任何协议的修改, 也已经成功地实现了一个人性化的大鼠系统9。因此, 这项研究描述了一个多才多艺的和有效的方法, 以产生充分的人类抗体, 可用于基础研究和免疫治疗。

<table><tbody><tr><td>HEK 293A cell line</td><td>Thermo Fisher scientific</td><td>R70507</td><td></td></tr><tr><td>DMEM (1X) Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco by life technologies</td><td>21969-035</td><td>(+) 4,5g/L D-Glucose&lt;br/&gt; 0,11g/L Sodium Pyruvate&lt;br/&gt; (-) L-Glutmine</td></tr><tr><td>RPMI medium1640 (1X)</td><td>Gibco by life technologies</td><td>31870-025</td><td></td></tr><tr><td>Bovine Serum Albumine (BSA)</td><td>PAA</td><td>K45-001</td><td></td></tr><tr><td>Nutridoma-SP</td><td>Roche</td><td>11011375001</td><td>100X Conc</td></tr><tr><td>PBS-Phosphate Buffered Saline (10X) pH 7,4</td><td>Ambion</td><td>AM9624</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid) 0,5M pH=8</td><td>Invitrogen by Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Fetal Bovine serum (FBS)</td><td>Dominique Dutscher</td><td>S1810-500</td><td></td></tr><tr><td>Ficoll - lymphocytes separation medium</td><td>EuroBio</td><td>CMSMSL01-01</td><td>density 1,0777+/-0,001</td></tr><tr><td>streptavidin R-phycoerythrin conjugate</td><td>Invitrogen by Life Technologies</td><td>S21388</td><td>premiun grade 1mg/ml contains 5mM sodium azide</td></tr><tr><td>Streptavidin, allophycocyanin conjugate</td><td>Invitrogen by thermoFisher scientific</td><td>S32362</td><td>1mg/ml&lt;br/&gt; 2mM azide premium grade</td></tr><tr><td>Brilliant violet 421 streptavidin</td><td>Biolegend</td><td>405225</td><td>conc : 0,5mg/ml</td></tr><tr><td>Anti-PE conjugated magnetic MicroBeads</td><td>Miltenyi Biotec</td><td>130-048-801</td><td></td></tr><tr><td>Anti-APC conjugated magnetic MicroBeads</td><td>Miltenyi Biotec</td><td>130-090-855</td><td></td></tr><tr><td>MidiMACs or QuadroMACS separotor</td><td>Miltenyi Biotec</td><td>130-042-302/130-090-976</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>CD3 BV510 BD horizon</td><td>BD Pharmingen / BD Biosciences</td><td>563109</td><td>Used dilution 1:20</td></tr><tr><td>CD19 FITC</td><td>BD Pharmingen / BD Biosciences</td><td>345788</td><td>Used dilution 1:20</td></tr><tr><td>CD14 PerCPCy5.5</td><td>BD Pharmingen / BD Biosciences</td><td>561116</td><td>Used dilution 1:50</td></tr><tr><td>CD16 PerCPCy5.5</td><td>BD Pharmingen / BD Biosciences</td><td>338440</td><td>Used dilution 1:50</td></tr><tr><td>7AAD</td><td>BD Pharmingen / BD Biosciences</td><td>51-68981E (559925)</td><td>Used dilution 1:1000</td></tr><tr><td>FACS ARIA III Cell Sorter Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>8-strip PCR tubes</td><td>Axygen</td><td>321-10-061</td><td></td></tr><tr><td>Racks for 96 microtubes</td><td>Dominique Dutscher</td><td>45476</td><td></td></tr><tr><td>RNAseOUT Ribonuclease Inhibitor (recombinant)</td><td>Invitrogen by thermoFisher scientific</td><td>10777-019</td><td>qty:5000U (40U/ul)</td></tr><tr><td>Distilled Water Dnase/Rnase Free</td><td>Gibco</td><td>10977-035</td><td></td></tr><tr><td>Oligod(T)18 mRNA Primer</td><td>New England BioLabs</td><td>S1316S</td><td>5.0 A260unit</td></tr><tr><td>Random hexamers</td><td>Invitrogen by thermoFisher scientific</td><td>N8080127</td><td>qty : 50uM, 5nmoles</td></tr><tr><td>Superscript III Reverse transcriptase</td><td>Invitrogen by thermoFisher scientific</td><td>18080-044</td><td>qty : 10000U (200U/ul)</td></tr><tr><td>GoTaq G2 Flexi DNA polymerase</td><td>Promega</td><td>M7805</td><td></td></tr><tr><td>dNTP Set, Molecular biology grade</td><td>Thermo Scientific</td><td>R0182</td><td>4*100umol</td></tr><tr><td>5LVH1</td><td>Eurofins</td><td>ACAGGTGCCCACT&lt;br/&gt; CCCAGGTGCAG</td><td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td></tr><tr><td>5LVH3</td><td>Eurofins</td><td>AAGGTGTCCAGTG&lt;br/&gt; TGARGTGCAG</td><td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td></tr><tr><td>5LVL4_6</td><td>Eurofins</td><td>CCCAGATGGGTCC&lt;br/&gt; TGTCCCAGGTGCAG</td><td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td></tr><tr><td>5LVH5</td><td>Eurofins</td><td>CAAGGAGTCTGTT&lt;br/&gt; CCGAGGTGCAG</td><td>First round of PCR - Amplification of heavy chains - Outer primers - Forward Prmers</td></tr><tr><td>3HuIgG_const_anti</td><td>Eurofins</td><td>TCTTGTCCACCTT&lt;br/&gt; GGTGTTGCT</td><td>First round of PCR - Amplification of heavy chains - Outer primers -Reverse primers for human Ig- Bacteria PCR screening</td></tr><tr><td>3CuCH1</td><td>Eurofins</td><td>GGGAATTCTCACA&lt;br/&gt; GGAGACGA</td><td>First round of PCR - Amplification of heavy chains - Outer primers -Reverse primers for human Ig</td></tr><tr><td>5AgeIVH1_5_7</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; GAGGTGCAGCTGGTGCAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH3</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCT&lt;br/&gt; GAGGTGCAGCTGGTGGAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH3_23</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCT&lt;br/&gt; GAGGTGCAGCTGTTGGAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH4</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; CAGGTGCAGCTGCAGGAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH4_34</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; CAGGTGCAGCTACAGCAGTG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH1_18</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; CAGGTTCAGCTGGTGCAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH1_24</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; CAGGTCCAGCTGGTACAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH3__9_30_33</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCT&lt;br/&gt; GAAGTGCAGCTGGTGGAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>5AgeIVH6_1</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCC&lt;br/&gt; CAGGTACAGCTGCAGCAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Forward primers</td></tr><tr><td>3SalIJH1_2_4_5</td><td>Eurofins</td><td>TGCGAAGTCGACG&lt;br/&gt; CTGAGGAGACGGTGACCAG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td></tr><tr><td>3SalIJH3</td><td>Eurofins</td><td>TGCGAAGTCGACG&lt;br/&gt; CTGAAGAGACGGTGACCATTG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td></tr><tr><td>3SalIJH6</td><td>Eurofins</td><td>TGCGAAGTCGACG&lt;br/&gt; CTGAGGAGACGGTGACCGTG</td><td>Second round of PCR - Amplification of heavy chains - Inner primers -Reverse primers</td></tr><tr><td>5&#039;LVk1_2</td><td>Eurofins</td><td>ATGAGGSTCCCYG&lt;br/&gt; CTCAGCTGCTGG</td><td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVk3</td><td>Eurofins</td><td>CTCTTCCTCCTGC&lt;br/&gt; TACTCTGGCTCCCAG</td><td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVk4</td><td>Eurofins</td><td>ATTTCTCTGTTGC&lt;br/&gt; TCTGGATCTCTG</td><td>First round of PCR - Amplification of light chains k - Outer primers -Forward primers</td></tr><tr><td>3&#039;Ck543_566</td><td>Eurofins</td><td>GTTTCTCGTAGTC&lt;br/&gt; TGCTTTGCTCA</td><td>First round of PCR - Amplification of light chains k - Outer primers -Reverse primers- Bacteria PCR screening</td></tr><tr><td>5&#039;AgeIVk1</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCT&lt;br/&gt; GACATCCAGATGACCCAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeIVk1_9_1&amp;ndash;13</td><td>Eurofins</td><td>TTGTGCTGCAACCGGTGTAC&lt;br/&gt; ATTCAGACATCCAGTTGACCCAGTCT</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeIVk1D_43_1_8</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTGT&lt;br/&gt; GCCATCCGGATGACCCAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeIVk2</td><td>Eurofins</td><td>CTGCAACCGGTGTACATGGG&lt;br/&gt; GATATTGTGATGACCCAGAC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeIVk2_28_2_30</td><td>Eurofins</td><td>CTGCAACCGGTGTACATGGG&lt;br/&gt; GATATTGTGATGACTCAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeVk3_11_3D_11</td><td>Eurofins</td><td>TTGTGCTGCAACCGGTGTAC&lt;br/&gt; ATTCAGAAATTGTGTTGACACAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeVk3_15_3D_15</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCA&lt;br/&gt; GAAATAGTGATGACGCAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeVk3_20_3D_20</td><td>Eurofins</td><td>TTGTGCTGCAACCGGTGTAC&lt;br/&gt; ATTCAGAAATTGTGTTGACGCAGTCT</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;AgeVk4_1</td><td>Eurofins</td><td>CTGCAACCGGTGTACATTCG&lt;br/&gt; GACATCGTGATGACCCAGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>3&#039;BsiWIJk1_2_4</td><td>Eurofins</td><td>GCCACCGTACGTT&lt;br/&gt; TGATYTCCACCTTGGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>3&#039;BsiWIJk3</td><td>Eurofins</td><td>GCCACCGTACGTT&lt;br/&gt; TGATATCCACTTTGGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>3&#039;BsiWIJk5</td><td>Eurofins</td><td>GCCACCGTACGTT&lt;br/&gt; TAATCTCCAGTCGTGTC</td><td>Second round of PCR - Amplification of light chains k - Inner primers -Forward primers</td></tr><tr><td>5&#039;LVl1</td><td>Eurofins</td><td>GGTCCTGGGCCCA&lt;br/&gt; GTCTGTGCTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVl2</td><td>Eurofins</td><td>GGTCCTGGGCCCA&lt;br/&gt; GTCTGCCCTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVl3</td><td>Eurofins</td><td>GCTCTGTGACCTC&lt;br/&gt; CTATGAGCTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVl4_5</td><td>Eurofins</td><td>GGTCTCTCTCSCA&lt;br/&gt; GCYTGTGCTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVl6</td><td>Eurofins</td><td>GTTCTTGGGCCAA&lt;br/&gt; TTTTATGCTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;LVl7</td><td>Eurofins</td><td>GGTCCAATTCYCA&lt;br/&gt; GGCTGTGGTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5LVl8</td><td>Eurofins</td><td>GAGTGGATTCTCA&lt;br/&gt; GACTGTGGTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>3&#039;Cl</td><td>Eurofins</td><td>CACCAGTGTGGCC&lt;br/&gt; TTGTTGGCTTG</td><td>First round of PCR - Amplification of light chains &amp;lambda; - Outer primers -Forward primers</td></tr><tr><td>5&#039;AgeIVl1</td><td>Eurofins</td><td>CTGCTACCGGTTCCTGGGCC&lt;br/&gt; CAGTCTGTGCTGACKCAG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>5&#039;AgeIVl2</td><td>Eurofins</td><td>CTGCTACCGGTTCCTGGGCC&lt;br/&gt; CAGTCTGCCCTGACTCAG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>5&#039;AgeIVl3</td><td>Eurofins</td><td>CTGCTACCGGTTCTGTGACC&lt;br/&gt; TCCTATGAGCTGACWCAG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>5&#039;AgeIVl4_5</td><td>Eurofins</td><td>CTGCTACCGGTTCTCTCTCS&lt;br/&gt; CAGCYTGTGCTGACTCA</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>5&#039;AgeIVl6</td><td>Eurofins</td><td>CTGCTACCGGTTCTTGGGCC&lt;br/&gt; AATTTTATGCTGACTCAG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>5&#039;AgeIVl8</td><td>Eurofins</td><td>CTGCTACCGGTTCCAATTCY&lt;br/&gt; CAGRCTGTGGTGACYCAG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -forward primers</td></tr><tr><td>3&#039;XhoICl</td><td>Eurofins</td><td>CTCCTCACTCGAG&lt;br/&gt; GGYGGGAACAGAGTG</td><td>Second round of PCR - Amplification of light chains &amp;lambda; - Inner primers -Reverse primers - Bacteria PCR screening</td></tr><tr><td>Ab-vec-sense</td><td>Eurofins</td><td>GCTTCGTTAGAAC&lt;br/&gt; GCGGCTAC</td><td>Bacteria PCR screening</td></tr><tr><td>QA Agarose-TM, Molecular Biology Grade</td><td>MP Bio</td><td>AGAH0500</td><td></td></tr><tr><td>NucleoFast 96 PCR Plate</td><td>Macherey Nagel</td><td>743.100.100</td><td></td></tr><tr><td>Enzyme Age I HF</td><td>New England Biolabs</td><td>R3552L</td><td>20000U/ml</td></tr><tr><td>Enzyme SalI HF</td><td>New England Biolabs</td><td>R3138L</td><td>20000U/ml</td></tr><tr><td>Enzyme Xho I</td><td>New England Biolabs</td><td>R0146L</td><td>20000U/ml</td></tr><tr><td>Enzyme BSIWI</td><td>New England Biolabs</td><td>R0553L</td><td>10000U/ml</td></tr><tr><td>HCg1 (Genbank accession number FJ475055)</td><td></td><td></td><td></td></tr><tr><td>LCk (Genbank accession number FJ475056 )</td><td></td><td></td><td></td></tr><tr><td>LCl (Genbank accession number FJ517647)</td><td></td><td></td><td></td></tr><tr><td>T4 DNA ligase</td><td>Invitrogen by thermoFisher scientific</td><td>15224.017</td><td>100U (1U/ul)</td></tr><tr><td>2X YT medium</td><td>Sigma Aldrich</td><td>Y1003-500ML</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>10835242001</td><td></td></tr><tr><td>LB (Luria Bertani) Broth (Lennox)</td><td>Sigma Aldrich</td><td>L3022-250G</td><td></td></tr><tr><td>Nucleospin Plasmid DNA, RNA and protein purification</td><td>Macherey Nagel</td><td>740588.250</td><td></td></tr><tr><td>Jet PEI DNA transfection reagent</td><td>PolyPlus</td><td>101-40</td><td></td></tr><tr><td>Flat bottom96-well plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>V-bottom 96-well plate</td><td>Nunc/Thermofisher</td><td>055142</td><td></td></tr><tr><td>Nunc easy 175 cm2 flasks</td><td>Nunc/Thermofisher</td><td>12-562-000</td><td></td></tr><tr><td>ELISA/ELISPOT coating buffer</td><td>eBiosciences</td><td>00-0044-59</td><td></td></tr><tr><td>Nunc maxisorp flat bottom 96 well ELISA plates</td><td>Nunc/Thermofisher</td><td>44-2404-21</td><td>high protein binding</td></tr><tr><td>Anti-human IgG Ab conjugated to horseradish peroxidase (HRP)</td><td>BD Pharmingen / BD Biosciences</td><td>55788</td><td></td></tr><tr><td>TMB substrate</td><td>BD Biosciences</td><td>555214</td><td></td></tr><tr><td>Streptavidin</td><td>Sigma</td><td>S0677</td><td></td></tr><tr><td>1 mL-HiTrap protein A HP column</td><td>GE Healthcare</td><td>17-0402-01</td><td></td></tr><tr><td>&amp;Auml;KTA FPLC</td><td>GE Healthcare</td><td>18190026</td><td></td></tr><tr><td>Superdex 200 10/300 GL column</td><td>GE Healthcare</td><td>17517501</td><td></td></tr><tr><td>NGC Quest 10 Plus Chromatography System</td><td>BioRad</td><td>7880003</td><td></td></tr></tbody></table>

generation of discriminative human monoclonal antibodies from rare antigen-specific b cells circulating in blood

单克隆抗体 (抗体) 是一种强有力的工具, 对基础研究和生物医学都很有用。当抗体需要区分高度相关的结构 (如、正常蛋白质和其突变版本) 时, 它们的高特异性是不可或缺的。目前产生这种歧视性抗体的方法包括广泛筛选多 Ab 生产的 B 细胞, 这既费钱又费时。本文提出了一种快速、成本效益高的方法, 从人血循环 B 淋巴细胞出发, 对人类抗体进行区分。这一策略的独创性是由于选择了特定的抗原结合 B 细胞结合所有其他细胞的反选择, 使用现成的外周血单个核细胞 (PBMC)。一旦分离出特定的 B 细胞, 使用单细胞逆转录-聚合酶链反应 (rt-pcr) 技术, 在人体内表达 cDNA (互补脱氧核糖核酸) 序列细胞.在短短的1月内, 有可能产生毫克的高度歧视性的人类抗体针对几乎任何所需的抗原自然检测的 B 细胞的剧目。

从 PBMC 从健康捐赠者开始, 这个项目提出了人类抗体的世代, 它认可肽 Pp65495 (Pp65, 从人巨细胞病毒) 提出由主要的组织相容性复合 i 类 (MHC i) 分子 HLA-A*0201 (HLA-A2)。这些抗体可以区分这一复杂和代表其他肽加载到同一 MHC I 分子的络合物。
PBMC 被 HLA-A2/Pp65-PE、HLA-A2/Pp65-APC 和 HLA-A2/MelA2-BV421 体染色, 如上述协议所述。聚丙烯?...

Watch this Scientific Journal Video about Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood at JoVE.com

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

我们描述了一种生产人类抗体特异性抗原的方法, 从人类血液中的稀有 B 细胞开始。这些天然抗体的产生是高效和快速的, 而获得的抗体可以区分高度相关的抗原。

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 ...

generation-discriminative-human-monoclonal-antibodies-from-rare

Research

JoVE Journal

Immunology and Infection

10.3K 次观看.  Université de Nantes. 我们描述了一种生产人类抗体特异性抗原的方法, 从人类血液中的稀有 B 细胞开始。这些天然抗体的产生是高效和快速的, 而获得的抗体可以区分高度相关的抗原。

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

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

科研

免疫与感染

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 ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

抗体生成:使用杂交瘤生产单克隆抗体

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

教育

JoVE Science Education

高级生物学

免疫学

Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Source: Nogales-Gadea, G. et al., <a href="https://app.jove.com/v/52830">Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Peripheral Blood ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Source: Devilder, M., et al. <a href="https://app.jove.com/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.