Name: generation of murine monoclonal antibodies by hybridoma technology
Uploaded: 2017-01-02T12:30:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

The authors acknowledge the German Federal Ministry of Education and Research (BMBF, Grant No: 03IPT7030X, 03IPT703A, and 03IP703) for funding our projects, "Artificial immune reactions," "Camelid antibodies," and "Antibody technologies." We thank Prof. Burkhard Micheel for proofreading the manuscript and for the helpful comments.

从我们的波茨坦大学（波茨坦，德国）繁殖群的BALB / c或NMRI小鼠（ 小家鼠 ）被用于生产单克隆抗体。动物工作是按照相关的国家和国际准则进行的。这项研究是经卫生部勃兰登堡环境，卫生和消费者保护（参考号码V3-2347-A16-4-2012）。 1.免疫缀合物的制备<ol><li>对抗原偶联到载体，通过氨基使用标准偶联方案，羧基或磺基的基团，例如通过戊二醛，N - （3-二甲基氨基丙基） - N -ethylcarbodiimide（EDC）或N - [Y-马来酰亚胺]琥珀酰亚胺酯（磺基GMBS）。 </li><li>比例为1:用于耦合（ 例如，用戊二醛）13，在1使用目标蛋白或肽和载体（卵清蛋白，牛血清白蛋白，或钥孔血蓝蛋白）。淡化目标PRotein或在磷酸盐缓冲盐水（PBS），并在50mM 碳酸氢钠缓冲液中的载体蛋白的肽。适应的体积和浓度，以必要的免疫接种的量。 <ol><li>混合这两种解决方案，并添加1.25％戊二醛。混合并孵育样品在室温（RT）下4小时。要选择的杂交瘤细胞，进行相同的免疫偶联物，但与另一种载体，以避免非特异性载体结合剂的选择。 </li></ol></li><li>通过用商业粗树脂（ 例如 ，交联葡聚糖G25）执行快速透析透析耦合样品。 <ol><li>穿孔的1.5ml反应小瓶的底部用中空针，并将其放置在一个15毫升管中。放玻璃棉在底部密封，并与300毫克粗树脂（0.5毫升的校准标记）的覆盖。 </li><li>小心，把1毫升的PBS滴加到1.5毫升反应管，并确保该玻璃棉仍然密封穿孔。让粗树脂小号井和离心机在500×g离心2分钟的列。一个这样的塔可以用于透析耦合样品的200微升。对于更高的产量，使更多的列。 </li><li>改管并删除耦合求解到膨胀粗树脂。离心再次在500 xg离心2分钟。从管中取出洗脱液，将其用于免疫。 </li></ol></li></ol> 2.动物免疫<ol><li>选择用于免疫两个或三个6-至12周龄的Balb / c小鼠。 </li><li>对于一个动物，准备150 - 在PBS 200微升免疫的200微克，并与100微升完全Freund&#39;s佐剂（CFA）混合。混合溶液具有短和细针3倍（0.9毫米直径的）上的1-mL注射器。腹膜内注射该溶液到小鼠。 </li><li>得到6-周后用相同量的免疫缀合物的加强注射，但是没有CFA。取血在7天后（出眶后窦）和准备工作再通过孵育血液3血清 - 在室温（RT）下4小时。 <ol><li>离心该混合物以3,000×g离心5分钟。采取血清上清液，并转移到一个新的试管中。在-20℃保存。弃沉淀。 </li></ol></li><li>分析通过酶联免疫吸附血清，如步骤3.对于融合描述的，选择具有最高血清效价的小鼠。 </li><li>在细胞融合的准备，加强与150鼠标 - 在PBS 300微升免疫的200微克无佐剂的融合前只有4天。 </li></ol> 3. ELISA <ol><li>制备具有5微克/毫升在PBS中的浓度的抗原溶液并转移50微升到每个ELISA孔96孔板的。 </li><li>在37℃或4℃的潮湿室中过夜孵育2小时的板。 </li><li>用自来水清洗板3次。在潮湿的通道阻塞，每孔80微升的PBS / 5％新生小牛血清的（NCS）的孔中，在室温1小时琥珀色。 5系列起动1:50:在1准备血清稀释。对于细胞培养的分析中，使用未稀释的培养物上清液。 </li><li>用自来水清洗板3次。吸移管50，每孔样品微升并在湿润室中在室温下孵育1小时。制备在1二次抗体溶液:5,000稀释缀合至辣根过氧化物酶（HRP）山羊抗小鼠IgG抗体。 </li><li>用自来水冲洗该板3次，并添加每孔的第二抗体溶液50微升。在室温下孵育在潮湿室中45分钟。 </li><li>准备一个新鲜的底物溶液。使为0.1M的NaH 2 PO 4和0.1％过氧化氢的储备溶液。取5份的NaH 2 PO 4，4份的过氧化氢，和用于新鲜底物溶液1份四甲基联苯胺。 <ol><li> 10次​​用自来水清洗板。吸移管50的新鲜底物溶液加入到孔中微升并孵育5 - 在10分钟黑暗。 </li></ol></li><li>停止与50微升1 MH 2 SO 4每口井的反应。使用板阅读器，测量在450nm处的光密度与在630nm的参考波长。 </li></ol> 4.骨髓瘤细胞培养制备<ol><li>从现在起，执行无菌的条件下工作。 </li><li>制备用于细胞培养新鲜培养基。补充500毫升的RPMI1640中，用10％胎牛血清（50毫升），2mM的L-谷氨酰胺（5毫升）中，50μM的巯基乙醇（5毫升）中。替代谷氨酰胺每7天。 </li><li>解冻鼠SP2 / 0细胞在温水中的小瓶。细胞溶液转移至10毫升新鲜培养基和离心机在200×g下10分钟。弃上清，悬浮颗粒在1毫升新鲜培养基。 </li><li>转移细胞在25-cm 2的烧瓶中并在培养箱中在37℃孵育它们和5％ 的 CO 2。扩大细胞两个75米2烧瓶BEF矿石融合。保存等分试样用于长期储存，如在步骤7中描述。 </li></ol> 5.准备饲养细胞的<ol><li>牺牲一个12周大的NMRI小鼠（根据国家和机构的指导方针; 例如，用CO 2吸入）。取下毛皮和清洁用70％乙醇的腹部。 </li><li>注入5毫升冰冷的RPMI培养基到腹部，按摩使用镊子腹部，并吸出饲养细胞悬浮液。放置细胞的一个空的50毫升管中。再次执行此步骤。 7至10毫升的饲养细胞悬浮液的应检索。 </li><li>通过加入90毫升全细胞培养基（RPMI 1640,10％胎牛血清，2mM谷氨酰胺，以及50μM的巯基乙醇）制备100毫升饲养细胞悬浮液。混合，并转移100μL/孔成10×96孔细胞培养板中。孵育所述板在37℃和5％CO 2在培养箱中过夜。 </li></ol> 6.细胞融合<ol><li>颈椎脱位或CO 2吸入牺牲免疫小鼠，并切除脾脏14。从心脏取额外血液并准备血清，如在步骤2.3所述，在ELISA中的阳性对照。 </li><li>通过细胞过滤网按压脾制备脾细胞悬浮液。填写多达20毫升全细胞培养基。收获在50毫升的管中的细胞通过离心分离（200 XG，10分钟，4℃）。弃去上清液。 </li><li>冲洗细胞培养烧瓶中，并通过离心收获的骨髓瘤细胞（200 xg离心，10分钟，4℃），弃上清。 </li><li>再次离心;重悬细胞沉淀在20ml平衡盐缓冲液（BSS）（pH为7.4的125mM的NaCl，5mM的氯化钾，4毫氯化钙 ，2.5mM的MgCl 2的，和5mM的Tris-HCl）。重复洗涤步骤三次。 </li><li>为了调整脾细胞:骨髓瘤细胞的比例，细胞计数第二洗涤步骤由杉木后ST重悬浮在1毫升的BSS缓冲液中沉淀。使1:10稀释并转移10微升至细胞计数室。计数细胞，并确定在1毫升的细胞浓度。 </li><li>调整中的BSS缓冲到三部分的脾细胞和单部分骨髓瘤细胞的细胞数在1毫升最终体积。取细胞悬浮液200微升，并将其与PEG8000的200微升（1克在4mL的BSS缓冲液中）混合。转移400μL成电穿孔杯中，直径为2毫米，并设置脉冲（600 - 650 V和25毫秒）。 <ol><li>脉冲后，孵育细胞在室温下在试管3分钟。除去细胞，并把它们滴加入Erlenmeyer烧瓶用100mL HAT培养基（RPMI 1640,10％FCS，2mM谷氨酰胺，50μM巯基乙醇，100μM的次黄嘌呤，5.8μM重氮丝氨酸，和16μM胸苷）。 </li><li>重复融合4次，直至1毫升批次花费。孵育3小时的Erlenmeyer烧瓶在37℃和5％CO 2的在孵化器。 </li></ol></li><li>电镀细胞饲养层上之前与次黄嘌呤，重氮丝氨酸和胸苷再次补充细胞悬浮液。加入100微升每孔的细胞悬浮液到饲养层板，并在37℃和5％的CO 2（在培养箱中）孵育所述板。阱内的最后浓度应为100μM次黄嘌呤，5.8μM重氮丝氨酸，和16微米的胸苷。 </li><li>为了使后7天克隆生长在板的各孔中，以确定是否井单 - 或多克隆:执行一个显微分析（10X物镜，10X目镜倍率）。单克隆细胞是从一个单细胞起源的细胞的一个簇。多克隆细胞是在一个孔中的细胞的多个群集。变更完成后，媒体7天，7天在HAT培养基中培养再次。 </li><li>更改HAT培养基，培养细胞的全细胞培养基。使用上清液进行的ELISA，如步骤3所述的正杂交瘤细胞必须在24孔板根据用于生产特异性抗体的测试扩展。如果细胞是多克隆的，有限的稀释必须执行（在步骤8中描述）。 </li></ol> 7.杂交瘤的冷冻保存<ol><li> Cryoconserve对于长期储存阳性单 - 和多克隆杂交瘤。收获来自24孔板的细胞通过离心&gt; 60％汇合（200 XG，10分钟，4℃）。 </li><li>重悬沉淀充分的细胞培养基的500微升，并将其转移到一个冻存管。加入500微升冷冻介质（RPMI 1640，20％FCS和15％二甲亚砜），混合它，并把它放在一个泡沫塑料箱或特殊冷冻箱。储存于-80℃3天。后3天，将冻存管可以转移到液氮长期贮存。 </li></ol> 8.限制多克隆杂交瘤稀释<ol><li>麦EA饲养层培养，如在第5步收获阳性多克隆细胞，被描述为在步骤7.1中描述。计数细胞，如步骤6.5中所述，并调节细胞数至10个细胞/ mL，用10毫升的96孔板的最终体积。添加100μL/孔到饲养层上。 </li><li>孵育7天后，在37℃和用显微镜分析所述克隆生长（倍率:10倍物镜，10X目镜）。使在ELISA培养物上清液，如在步骤3中描述，并确定为大量培养适当的单克隆杂交瘤。 </li></ol> 9.大众文化和单克隆抗体的纯化<ol><li>转移用适当的ELISA信号的单克隆杂交到25 平方厘米或75 平方厘米培养瓶中，并收集到500毫升的培养上清。测试培养每周特异性抗体的生产和保存3 - 每杂交瘤细胞5等份用于长期储存，如在步骤7中所述。 </li><li&gt;离心4900 xg离心培养上清液，4℃15分钟，通过0.45微米的过滤器过滤。混合在一个3结合缓冲液（4M氯化钠和2M甘氨酸的NaOH，pH值8.9）的上清液:1的比例。 </li><li>通过用洗涤缓冲液洗涤制备蛋白A柱（稀释结合缓冲液1:3在蒸馏水中2 O）。来自步骤9.2在塔传上清，收集流过。洗脱结合的抗体用0.1M柠檬酸盐，pH为3.5，用500微升的Tris-HCl的，pH为9.0，立即中和pH。 </li><li>表征通过SDS-PAGE，蛋白质印迹，和ELISA洗脱抗体，并验证它的最终应用。 <ol><li>对于SDS PAGE，取3 - 5％车道（20μL样品）纯化的抗体溶液微克，加入5μL的4倍SDS样品上样缓冲液（8％SDS，20％2-巯基乙醇，40％甘油，和0.015 ％溴酚，还原的），并加热，在95℃下的探针5分钟。作为对照，使用相同的样品与纯化同型但不相关的特异性ntibody。 </li><li>装入样品在12.5％SDS凝胶，将未染色的蛋白梯5μL在另外的车道，并通过电泳将它们分开。染色考马斯亮蓝凝胶并记录结果。一个基本的SDS协议由拉姆力于1970年15成立。 </li></ol></li></ol>

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G., Wagner, D., Heilmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+antibody+production+by+intrarectal+immunization+of+mice+using+recombinant+major+capsid+protein+of+hamster+polyomavirus.">IgA antibody production by intrarectal immunization of mice using recombinant major capsid protein of hamster polyomavirus.</a> Eur J Microbiol Immunol. 2 (3), 231-238 (2012).</li><li>Listek, M., Micheel, B., Hanack, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomoleküle+freisetzende+zelle+und+deren+selektion+mittels+eines+oberflächenproteins.">Biomoleküle freisetzende zelle und deren selektion mittels eines oberflächenproteins.</a> neweramabs GmbH. , (2014).</li></ol>

The authors Pamela Holzl&#246;hner and Katja Hanack are the co-founders of new/era/mabs GmbH, a company that offers the customized generation of monoclonal antibodies.

通过杂交瘤技术的单克隆抗体的产生需要的强烈和详细的表位分析，特别是对最终的应用中，当该抗体应当识别目标。这通常是由用户低估并导致弱表演抗体。融合过程总是随机的，这意味着特异杂交的结果在很大程度上取决于在该点的细胞比例和活力。有限稀释后，细胞是非常不稳定的，需要的细胞生长和抗体生产的严格监控。这些参数应排除故障的方法时，应仔细分析。 <p class="jove_content"...

在这个协议中提出的杂交瘤技术最早是由和Milstein 1在1975年描述的，除了一些技术改进，主要的程序没有显着在过去的40年2变化。该协议的目的是说明一个更合适的免疫策略，对于单克隆抗体的产生的标准方法，以及用于验证方法（ELISA）的例子。 抗体是难以置信的工具和向广泛的技术方法，如流式细胞术，磁细胞分选，或免疫荧光，以及用于疾病监测和治疗3的诊断和治疗方案。对于所需靶的单克隆抗体的商业供应是通过在24个不同的网络数据库的存在下，用抗体或抗体相关的产品4的几乎无数数量证明。 2015年，一ntibody分子的国际讨论5-7的部分原因是正确的验证和市售抗体的表征严重的问题。 它可能是困难和昂贵找到靶向抗原的特异性抗体，并且通常，它们不具有所需要的亲合性或特异性。虽然产生的抗体仍然是耗时且需要熟练人员开发和验证的抗体，单独地产生抗体可能比购买更好。 由于这样的事实:抗体的产生是耗费时间的，并且需要经验，为生产结合分子的替代方法被开发来克服这些问题。最常用的一种方法是通过噬菌体展示的重组生产单链抗体。用于可变结合区的基因是从细胞中提取，并用噬菌体的涂布蛋白质相结合。在S英格尔链然后噬菌体的表面上表达，并在几个平移步骤8进行筛选。生产的单链抗体的有点快，但它也需要一个熟练的实验者。一些重组单链抗体的缺点是稳定性差并用于体外诊断方面的缺乏适合的。在大多数诊断测试的Fc受体进行检测是必要的，这需要被添加到一个重组单链抗体之后。再次，这是费时和甚至比杂交瘤技术更加复杂。在体外诊断，全长单克隆小鼠和兔抗体已被证明是最好的选择。 所述免疫缀合物的设计:一个在产生单克隆抗体的主要步骤必须在实验室中的工作开始之前完成。需要解决的问题是:什么是目标在最终APPLICAT物理组合物离子，并且该基质是本？其中浓度将目标必须在应用程序？什么是最终的应用，什么是该抗体必须满足的要求？ 总是考虑到，如果使用线性肽片段，它也有在所选择的目标的最后的表位是线性;否则，将抗体不结合。当然，独立的筛选方法的，抗体可被选择为识别在不同的应用不同的抗原的格式，但是这必须被非常精确地验证。这就是为什么抗体的开发和验证是这样雄心勃勃的过程的原因。 用于免疫的抗原格式的选择是对抗体发展的根本，并确定该过程的成功或失败。一旦小鼠表达相关的抗体滴度，所述脾细胞分离并用骨髓瘤细胞融合。最常见的骨髓瘤细胞系为鼠单克隆抗体的开发是X63-银8.653 9和来自Balb / c小鼠品系的Sp2 / 0-银14 10。将细胞从一个恶性B细胞淋巴瘤下降和被选择，因为他们不分泌任何自己重链或轻链。 1（脾细胞与骨髓瘤细胞）:细胞可以1:10和10之间适于比率。在这个协议中，将细胞适应于3:1的比率和由聚乙二醇（PEG）和电融合融合，根据Stoicheva和辉11。 B细胞和骨髓瘤细胞的融合是一个随机过程。因此，可以产生两个B淋巴细胞或两个骨髓瘤细胞的杂交体，但这些杂种将无法用于在培养很长时间存活。细胞经历一次黄嘌呤，氨基喋呤，和胸苷（HAT）的选择，其中只有融合杂交瘤细胞可存活由于使用嘧啶合成的从头合成途径的可能性。对于周一的产生oclonal抗体，它是必要的，以获得从一个母细胞始发的细胞系。的单克隆通过限制稀释技术和细胞生长的显微分析确保。杂交瘤培养上清液筛选特异性抗体的生产，主要是通过ELISA或流式细胞术，和最好的粘合剂来选择。大量培养和纯化后，该抗体分子可以最后被表征和验证为所需要的应用程序。

<table><tbody><tr><td>glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&lt;/em&gt;-ethylcarbodiimide (EDC)</td><td>Sigma Aldrich</td><td>39391-10ML</td><td></td></tr><tr><td>Sulfo-GMBS</td><td>Perbio Science Germany</td><td>22324</td><td></td></tr><tr><td>ovalbumin</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>keyhole limpet hemocyanin</td><td>Sigma Aldrich</td><td>H8283</td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Biochrom GmbH</td><td>P91015</td><td></td></tr><tr><td>reaction vials, 1.5 mL</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>CNT2.1</td><td></td></tr><tr><td>hollow needle</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>C724.1</td><td></td></tr><tr><td>glass wool</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6574.1</td><td></td></tr><tr><td>Sephadex G25 coarse</td><td>Sigma Aldrich</td><td>GE-17-0034-02</td><td></td></tr><tr><td>Freund&amp;acute;s adjuvant, complete</td><td>Sigma Aldrich</td><td>F5881-10ML</td><td></td></tr><tr><td>ELISA plates, 96 well</td><td>Greiner bio-one</td><td>655101</td><td></td></tr><tr><td>neonatal calf serum</td><td>Biochrom GmbH</td><td>S1025</td><td></td></tr><tr><td>TipOne Tips 1,000 &amp;micro;L</td><td>Starlab</td><td>S1111-2021</td><td></td></tr><tr><td>Pipette tips 200 &amp;micro;L</td><td>Greiner bio-one</td><td>739291</td><td></td></tr><tr><td>HRP-conjugated goat-anti-mouse IgG antibody</td><td>Dianova</td><td>115-035-003</td><td></td></tr><tr><td>tetramethylbenzidine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6350.2</td><td></td></tr><tr><td>Natriumdihydrogenphosphat</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K300.2</td><td></td></tr><tr><td>peroxide/urea</td><td></td><td></td><td></td></tr><tr><td>sulphuric acid</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4623.3</td><td></td></tr><tr><td>RPMI 1640</td><td>Life technologies GmbH</td><td>31870074</td><td></td></tr><tr><td>L-glutamine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>HN08.2</td><td></td></tr><tr><td>beta-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270106</td><td></td></tr><tr><td>TC-flask 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V025</td><td></td></tr><tr><td>TC-flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V075</td><td></td></tr><tr><td>ethanol, 96%</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>P075.1</td><td></td></tr><tr><td>cell strainer</td><td>VWR international</td><td>734-0002</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Biochrom GmbH</td><td>P91050</td><td></td></tr><tr><td>PEG 8000</td><td>Sigma Aldrich</td><td>1546605</td><td></td></tr><tr><td>electroporation cuvette, 2 mm</td><td>Biodeal Handelsvertretung Edelmann e.K.</td><td>EKL2,25</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma Aldrich</td><td>H9636-25G</td><td></td></tr><tr><td>azaserine</td><td>Sigma Aldrich</td><td>A4142</td><td></td></tr><tr><td>thymidine</td><td>USB Europa GmbH</td><td>22305 1 GM</td><td></td></tr><tr><td>TC-plates 96 well</td><td>Biochrom GmbH</td><td>P92696</td><td></td></tr><tr><td>TC-plates 24 well</td><td>Biochrom GmbH</td><td>P92424</td><td></td></tr><tr><td>cryotubes, 1 mL</td><td>Sigma Aldrich</td><td>V7384-1CS</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4720.1</td><td></td></tr><tr><td>protein A sepharose</td><td>Sigma Aldrich</td><td>P3391-1G</td><td></td></tr><tr><td>SDS sample loading buffer, Roti-Load 1</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K929.1</td><td></td></tr><tr><td>unstained protein ladder</td><td>BioRad Laboratories</td><td>161-0363</td><td></td></tr><tr><td>comassie brilliant blue R-250</td><td>BioRad Laboratories</td><td>161-0406</td><td></td></tr></tbody></table>

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.

图1显示了抗原特异性血清滴度为在实验室中进行的一次免疫的一个例子。相比5,000,000一个幼稚小鼠的血清:在该图中，免疫血清A和B从1:50到1滴定。抗原涂布在固相上，并用一个POD缀合的山羊抗小鼠Ig抗体进行检测的特异性抗体。相比于幼稚小鼠的免疫的小鼠的两个血清呈显著更高的抗原特异性滴度。以1:1的稀释度的信号:5000足以上移动到下一个步骤，所述细胞...

Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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-murine-monoclonal-antibodies-by-hybridoma-technology

Research

JoVE Journal

Immunology and Infection

41.5K 次观看.  University of Potsdam. 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. 

视频: Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

单克隆抗体的反对消极行政 H。 capsulatum和其他真菌病原体

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

科研

免疫与感染

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 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 ...

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 ...

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

猪肠粘膜上皮中靶向氨基肽酶N的单克隆抗体的生产

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

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, ...

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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 Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Source: Xia, P., et al. <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.

一种用于生产针对金属肽酶的单克隆抗体的杂交瘤技术

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

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.

一种从抗原特异性 B 细胞生成单克隆抗体的技术

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges K&#246;hler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques &#8212; electroporation, polyethylene glycol (PEG) mediated fusion, and the use of fusogenic viruses &#8212; generally produce hybridomas at a very low frequency. Fusion products thus have a high ratio of self-fused B-cells and myeloma cells, or unfused cells, with a low number of hybrid cells.
The hypoxanthine-aminopterin-thymidine (HAT) medium is used to select hybridomas by selectively allowing their growth. The aminopterin blocks the default nucleotide synthesis in cells. However, cells can utilize hypoxanthine and thymidine from the medium to synthesize nucleotides via the salvage pathway.
Myeloma cells deficient in the HGPRT enzyme cannot synthesize nucleotides via this pathway and thus, do not grow in HAT medium. Although B-cells produce functional HGPRT enzymes, they cannot divide indefinitely. Thus, only the hybrid cells with functional HGPT from the B-cells and immortality from the myeloma line can grow on the HAT selection medium. Such a culture of hybrid cells is called a hybridoma, which can be screened for monoclonal antibody production.
Hybridoma Screening
Hybridoma cultures from the HAT selection are plated onto a 96-well plate; each well containing only one cell. Using Enzyme-linked Immunosorbent Assay (ELISA), each cell is screened for the production of antibodies specific to the epitope of interest. Cells that test positive are then selected and grown in a larger culture vessel to establish hybridoma cell lines. These cell lines are a permanent source of unlimited monoclonal antibodies. Thus hybridoma technology is a convenient and cost-effective method for mass production of monoclonal antibodies.

杂交瘤技术

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or ...