这项工作是由卫生（R01 GM092993，R01 NS048357和R21 NS073684）全国学院的资金支持，美国国家科学基金会（CAREER奖），明尼苏达州合作奖的生物技术和医学基因组学，国家多发性硬化症协会（CA1060A）和梅奥诊所中心的临床和转化科学（CCATS）。作者承认从阿普尔鲍姆，希尔顿，彼得森和桑福德基金会，月球和玛丽莲公园出任董事基金和McNeilus家人的支持。

Patents for antibodies that promote remyelination and CNS repair are issued and owned by Mayo Clinic. Authors have a potential conflict of interest.

人天然IgM抗体正在呼吁免疫疗法的候选人，并已显示出对各种疾病的治疗包括癌症和中枢神经系统疾病1-7治疗潜力。有利的是，这些抗体不会引起免疫反应，其中的中和抗体的产生显着减少的有效治疗剂量和疗效。重要的是，具有治疗潜力的所有抗体一直是IgM同种型的并且属于NAB剧目3-5,8,9,37,40,42-45。为潜在的临床应用的IgM抗体的一个主要障碍是其抗原，它们在许多情况下，未确定的识别。对于IgG抗体采用标准技术通常并不适用于识别的IgM的抗原。 这个协议描述PSA-NCAM的识别作为抗原的再生人IgM抗体HIgM12，有效的动物模型MS的和ALS 15-18。所采用的方法主要适用于特定Vκ轻链VκI，VκIII和VκIV和鼠标与抗体轻VκI所有连锁人类抗体，而不论该抗体的同种型。 在这个协议中最关键的一步是在亲和层析应用中使用IgM抗体。更具体地，需要成功的抗体作为下拉剂免疫沉淀来分离或富集抗原出复杂的组织或细胞培养裂解物。富集抗原馏分可以进行比较，以从同种型对照抗体免疫沉淀，并随后分析通过质谱法的差异。一个基本要求或该步骤的限制是正确的抗体Vκ轻链和主机（人，小鼠）的存在下，使抗体结合到蛋白L琼脂糖。结合到琼脂糖甘露聚糖结合蛋白的用途（也卡勒Ð甘露糖结合凝集素），而不是蛋白质L琼脂糖是免疫沉淀的IgM没有具体Vκ轻链的潜在替代。然而，如由阿诺德等人 35所述，结合抗原的IgM抗体不结合甘露聚糖结合蛋白，作为目标聚糖似乎变得不可访问一旦IgM抗体已结合至其抗原。基于这些发现甘露聚糖结合蛋白，不能使用作为结合基质免疫沉淀负载抗原的IgM的分子35。其他潜在的替代品可能是利用二次琼脂糖结合的抗IgM IgG抗体46,47或使用表面活化磁珠48。针对的IgM IgG抗体可能是化学交联琼脂糖A或琼脂糖的G以与感兴趣的抗原一起减少洗脱抗体的程度。 IgM抗体免疫沉淀的不同种类之间的适当的比较就成功识别抗原的我因为大多数免疫沉淀进行隔离或耗尽的IgM没有在他们的抗原进一步加息很难。此外，使用的IgM免疫沉淀主要用来确认用抗体暴露于纯化的抗原49的已知的或预期的单一抗原。相比于原料50时免疫沉淀血清的IgM分子- （15％10）针对人的IgM琼脂糖偶联的IgG的一个缺点是一个非常低的产率。与此相反，表面活化磁珠成功地应用于不同的同种型包括IgM免疫沉淀瘙痒相关纤维48的抗体。此方法并不限于特定卡伯（Vκ）或lambda（λ）链或特定宿主，并且可以基本上扩大在免疫沉淀中使用IgM抗体的频谱。 在协议中的另一个重要步骤是该抗体的以用作DET能力阿拉斯在Western印迹或其他筛选平台抗体（初级抗体）。成功的抗体必须定位它们的抗原以足够高的亲和力，以允许抗原在非离子型或可能离子洗涤剂的存在下的结合。高亲和力抗体是亲和熟化IgG抗体中常见，但在IgM同种型，这就是为什么有相对少的市售IgM抗体作为生化设置检测剂的原因之一的抗体中不常见的。高亲和力抗体的结合是对分子的抗原的免疫沉淀反应和Western印迹的一个要求。降低洗涤剂浓度或完全不存在于IP缓冲液和裂解缓冲液的洗涤剂允许抗原通过其的Fab结构域由低亲和力抗体定位。相反，抗体的经由其Fc部分靶向蛋白L琼脂糖能力基本上不同种型对照中受影响的范围内不同的洗涤剂concen的trations。然而，在裂解缓冲液和IP缓冲器低洗涤剂浓度防止细胞膜破坏和特定分子的分离。同样地，在Western印迹洗涤缓冲液低洗涤剂浓度（ 例如，PBS-T）允许抗原低亲和力抗体的结合，但在同一时间增加的非特异性结合，因此不是一种选择。 抗原蛋白质核的存在是该方法的另一要求。用翻译后修饰（ 例如 ，糖蛋白，脂蛋白）和未修饰的蛋白质，但并非唯一的脂质或碳水化合物的蛋白质，是在Western印迹检测到。这是不可能的，以免疫沉淀从组织或细胞裂解物的脂质（ 例如，鞘脂）在洗涤剂中的存在或不存在。洗涤剂和脂质的物理化学性质是太相似，以允许选择性脂质 - 抗体相互作用，而在同一时间清净剂是必不可少的扰乱为了膜可以允许特定分子的选择性分离。据我们所知，免疫沉淀仅仅包括碳水化合物，在不存在的蛋白质核心的，还没有被报道。因为IgM抗体频繁目标碳水化合物和糖脂，这是没有必然联系的蛋白质单元这一点变得尤其重要。其他色谱技术都需要分离和免疫学鉴定脂质和碳水化合物在没有蛋白质核的。例如，在TLC板（免疫TLC）与随后抗体检测细胞脂质的薄层色谱法（TLC）可以实现51,52。 其他抗PSA抗体已在以往的研究和结果进行了描述比较了HIgM12以及市售抗PSA的IgM（克隆2-2B）。在PSA领域中最常用的抗体可作为小鼠monoc IgG抗体（克隆735）lonal或兔多克隆抗体53。此抗PSA IgG抗体检测的PSA上SynCAM和NCAM上不同的CNS细胞类型，包括小胶质细胞41。与此相反的抗PSA IgG抗体，人IgM抗体HIgM12，HIgM42和抗PSA小鼠IgM（克隆2-2B）无法定位到SynCAM PSA（但NCAM）在小鼠各种胚胎和出生后早期阶段，非唾液酸SynCAM是容易检测15。所使用的IgM抗体也不能检测在小神经胶质细胞（ 图3 + 4）的PSA。相比具有潜在的较低的亲和力相比，抗PSA抗体的IgM抗体的结合组合PSA-NCAM水平抗体同种型之间观察到的差异的可能解释可以是低的PSA SynCAM水平。为了检验这一假设，我们与大量SynCAM目前的执行中NCAM KO动物免疫沉淀在胚胎阶段E17和使用HIgM12作为"下拉代理"&lt;SUP&gt; 15。在E17 WT同窝控件HIgM12免疫沉淀PSA-NCAM到的程度，即显示出的"拉下"PSA-NCAM相比通过使用洗脱抗原随后印迹光密度检测出的的IgM重链相似的强度。这一结果表明，至少HIgM12有足够的亲和力，它的目标PSA。在对比WT同窝控制HIgM12没有检测附着到SynCAM在NCAM KO动物的PSA。相同的结果是使用人类抗PSA的IgM HIgM42在免疫沉淀得到的。 HIgM12和HIgM42，以及小鼠抗 - PSA IgM抗体（克隆2-2B）无法定位在胚胎和出生后的发育阶段从​​WT和NCAM KO动物印迹上SynCAM粘合剂。定所需HIgM12的低量（0.1微克/毫升），以特异性检测附着到NCAM在使用少量CNS组织（每孔0.1微克CNS组织）的Western印迹的PSA 15这似乎是不可能的，低亲和力单独是负责三种不同的方法完全缺乏PSA-SynCAM检测由HIgM12。 我们的结论是有抗PSA IgM抗体和频频发布抗PSA IgG抗体（克隆735）之间的显著差异。为什么市售抗PSA IgM抗体未在过去更频繁地使用，以确认与antiPSA IgG抗体735所获得的结果，因为HIgM12已经在不同的疾病模型已经证明效力，这是特别有趣的，目前尚不清楚。而其它抗PSA IgM抗体可以具有类似的治疗效果仍然不清楚的抗PSA IgG抗体（克隆735）是否具有在MS和ALS模型类似的治疗效果。 简言之，此处所描述的方法主要被用于识别再生人抗体HIgM12的抗原，以支持用于MS的潜在的临床试验和其他可能的神经变性疾病。蚁的识别为具有生物活性的抗体igens是了解其作用机理是必不可少的步骤。这成为在目前正在为在人体试验安全性和有效性进行测试大量的单克隆抗体的情况下特别相关。而临床测试IgM抗体迄今为止的数目是小的，最近在杂交瘤技术，越来越多的研究突出该抗体类1-10,37,40,42-45的治疗潜力一起进步促使新的或开发改性方法适用于鉴定非蛋白质的IgM抗原。

的IgM同种型的抗体表现出对各种疾病的治疗包括癌症和CNS障碍1-7大的治疗潜力。该Vollmers"组确定癌症患者大量抗体作为肿瘤特异性标志物，或者能够通过诱导凋亡途径4,8,9杀死恶性细胞活跃疗法的潜在用途。有趣的是，具有治疗潜力的所有确定抗体是IgM同种型的和属于该组的"天然抗体"（NAb的）的。 同样地，罗德里格兹的组识别鼠标和在多发性硬化症（MS）的模型刺激髓鞘慢性脱髓鞘脊髓病变的人抗体。相同的具有抗癌作用的抗体，所有的髓鞘再生，促进抗体的NAb和IgM同种型1,6,7,10的。对于大多数鉴定的IgM精确的抗原仍然undetermined包括MS患者11 I期临床试验的人的髓鞘再生，促进抗体rHIgM22，目前在阶段。尽管在脂类和碳水化合物研究的无论是在学术环境中，并与工业11合伙领域的专家多次努力，试图确定rHIgM22的抗原都没有成功。的用于识别IgG抗体的抗原与IgM抗体工作的标准技术故障标识关键需要改进特异于这些抗体，最有可能的目标碳水化合物或脂质抗原的方法。 这份手稿的重点是人的再生抗体HIgM12以及用于识别其抗原的实验程序。抗体HIgM12从患者瓦尔登斯特伦巨球蛋白血症鉴定，刺激体外 12-14神经突增生和目标连接到神经细胞粘附分子（PSA-NCA聚唾液酸（PSA）的M）15,16。 HIgM12延伸在ALS 17的小鼠模型中的寿命，并提高在Theiler鼠脑脊髓炎病毒（TMEV） -感染的小鼠的功能结果。具体地说，HIgM12刺激中小直径脊髓轴突8周慢性脱髓鞘小鼠和增加号码自发的水平和垂直运动活性后腹腔内的一个单一的，低剂量注射抗体18。 神经细胞粘附分子（NCAM）是免疫球蛋白（Ig）的神经元，神经胶质，骨骼肌，和自然杀伤细胞19-25的细胞表面上超家族表达的糖蛋白。三大NCAM亚型称为NCAM180，NCAM140和NCAM120，是一个主要的转录物的可变剪接变体，只有在其细胞质区域发生变化。内的中枢神经系统，NCAM是主要的唾液酸分子（&gt; 95％），长的，带负电荷的唾液酸的均聚物。多聚一CID具有n&gt; 10被称为PSA但较短的寡聚结构存在，同时也是生物相关。在中枢神经系统表达其他唾液酸蛋白SynCAM1 26，神经毡蛋白-2（NRP-2）和27,28钠通道亚基25（综述见29）。 这里所描述的方法允许含特定卡帕（Vκ）轻链（对于人抗体和VκI轻链为小鼠抗体VκI，VκIII或VκIV轻链）人和小鼠免疫球蛋白的抗原识别而不管抗体的同种型（ 例如，IgG抗体，IgM抗体的，IGA，IgD的或IgE）。这种限制是基于使用蛋白L琼脂糖的用于与IgM同种型的抗体免疫沉淀。替代策略可以包括甘露糖结合凝集素和共价连接到琼脂糖珠次级IgG抗IgM抗体，其可以扩大该方法的适用性更IgM抗体增量泸定那些拉姆达（λ）轻链（见讨论）。相比IgM抗体λ健康个体衍生轻链的IgM血清轻Vκ链的比率是1.5:1 30。 基于此处用来分离，富集和免疫检测特定分子31的色谱方法，都需要所有抗原包括至少一种小的蛋白质结构域。该抗体的特异性结合的表位可以是内部或蛋白质结构域（ 例如 ，在糖蛋白，脂蛋白）的外部。用于识别特定抗原HIgM12，各自以缩​​小潜在候选的列表中最初的生化步骤，是在该方法中最关键的步骤。细胞类型特异性制剂和基于细胞形态学-表征为神经胶质细胞被描述，但该方法可以外推到容纳内或中枢神经系统以外的其它类型的细胞。 目前迫切需要开发适用于数量的增加，其中所述抗体的目标是碳水化合物或脂质结构IgM抗体与特别是在这些情况下，对于不同的人类疾病的治疗潜力（多数的IgM抗原）的新的或修改的技术。

<table><tbody><tr><td>DMEM</td><td>Fisher Scientific</td><td>MT-10-013-CVRF</td><td>High glucose, with glutamine, with sodium pyruvate, 500 ml</td></tr><tr><td>DMEM/F-12, HEPES</td><td>Life Technologies</td><td>11330-032</td><td>500 ml, L-glutamine, sodium pyruvate, high glucose</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td>10,000 U/ml, 100 ml</td></tr><tr><td>N-2 Supplement (100x)</td><td>Life Technologies</td><td>17502-048</td><td>5 ml</td></tr><tr><td>B-27 Supplement (50x)</td><td>Life Technologies</td><td>17504-044</td><td>serum free, 10 ml</td></tr><tr><td>Fetal Bovine Serum - Optima</td><td>Atlanta Biologicals</td><td>S12450</td><td>500 ml</td></tr><tr><td>Sterile water for irrigation</td><td>Baxter Healthcare</td><td>#2F7114</td><td>USP-1,000 ml, 12/CA</td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7886-50MG</td><td>D-Lys-(D-Lys)n-D-Lys &amp;middot; xHBr, Molecular Weight 30,000-70,000 g/mol, 50 mg</td></tr><tr><td>bovine serum albumin fraction V</td><td>Sigma</td><td>A-3294-100G</td><td>heat shock fraction, protease free, pH 7,&amp;nbsp; purity 98%, 100 g</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G5767-500G</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, Molecular Weight: 180.16 g/mol, ACS reagent, 500 g</td></tr><tr><td>Trypsin from bovine pancreas</td><td>Sigma</td><td>T9935-100mg</td><td>essentially salt-free, lyophilized powder, 9,000 BAEE units/mg protein</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D5025-150KU</td><td>Type IV, lyophilized powder, 2,000 Kunitz units/mg protein</td></tr><tr><td>Recombinant Rat FGF basic Protein</td><td>R&amp;amp;D systems</td><td>3339-FB-025</td><td>BSA as a carrier protein, 25 &amp;#181;g, lyophilized, &gt; 95%, 16.2 kDa</td></tr><tr><td>Recombinant Rat PDGF-AA Protein</td><td>R&amp;amp;D systems</td><td>1055-AA-50</td><td>carrier free, 50 &amp;#181;g, lyophilized, &gt; 97%, &lt;em&gt;E. coli&lt;/em&gt;-derived, 12.5 kDa</td></tr><tr><td>Neurobasal-A</td><td>Life Technologies</td><td>10888-022</td><td>500 ml, No Glutamine, No Aspartic Acid, No Glutamic Acid</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td>crystalline, 1 kg, reagent grade, Molecular Weight 30.03 g/mol (as monomer)</td></tr><tr><td>Tris[hydroxymethyl]aminomethane (Tris bas</td><td>Biorad</td><td>#1610719</td><td>1 kg, 99.8% pure, powder</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Biorad</td><td>#1610302</td><td>1 kg, powder</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP-381-5</td><td>C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;NO&lt;sub&gt;2&lt;/sub&gt;, Molecular weight: 75.07 g/mol, White crystalline Powder, 5 kg</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653-5KG</td><td>NaCl, Molecular Weight: 58.44 g/mol, 5 kg</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S0751-500G</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Molecular Weight: 119.98 g/mol, 500 g</td></tr><tr><td>Phosphate-buffered Solution 1x (PBS)</td><td>Cellgro</td><td>MT-21-040-CV</td><td>Without Calcium and Magnesium, 6 x 500 ml</td></tr><tr><td>Falcon 60 mm TC-Treated Cell Culture Dish</td><td>Corning Life Sciences</td><td>#353002</td><td>60 mm Cell Culture Dish, TC-treated polystyrene, 20/pack, sterile</td></tr><tr><td>Falcon 60 mm x 15 mm Petri dish</td><td>Corning Life Sciences</td><td>#351007</td><td>Not TC-Treated Bacteriological Petri Dish, 20/Pack</td></tr><tr><td>6 well cell culture plates</td><td>Corning Costar</td><td>CLS3516</td><td>6 well, flat bottom (Individually wrapped), gamma-irradiated, growth area 9.5 c</td></tr><tr><td>75 cm&amp;sup2; tissue culture flask</td><td>Corning Life Sciences</td><td>430720U</td><td>75 cm&amp;sup2; U-Shaped Canted Neck Cell Culture Flask with Plug Seal Cap</td></tr><tr><td>Pierce&amp;trade; Protein L Plus Agarose</td><td>ThermoFisher Scientific</td><td>#20520</td><td>2 ml, crosslinked 6% beaded agarose (CL-6B), binding capacity: 10 to 20 mg IgG</td></tr><tr><td>4 - 20% Mini-PROTEAN TGX Precast Gel</td><td>Biorad</td><td>456-1094</td><td>10-well, 50 &amp;micro;l, for use with Mini-PROTEAN electrophoresis cells</td></tr><tr><td>Immobilon-P Membrane</td><td>Millipore</td><td>IPVH00010</td><td>PVDF, 0.45 &amp;micro;m, 26.5 cm x 3.75 m roll</td></tr><tr><td>Anti-Polysialic Acid-NCAM Antibody, clone</td><td>Millipore</td><td>MAB5324</td><td>50 microliters, Host: mouse, monoclonal IgM</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Biorad</td><td>#1610747</td><td>10 ml, 4x premixed protein sample buffer</td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>#1610710</td><td>25 ml, 98 % pure, 14.2 M</td></tr><tr><td>Endoneuraminidase-N (Endo-N)</td><td>ABC Scientific</td><td>ABC0020</td><td>50 microliters, 200 &amp;micro;g/ml, 3,500 U/mg</td></tr><tr><td>25 mm glass coverslips</td><td>Fisher Scientific</td><td>12-545-102</td><td>Circles No. 1; Thickness: 0.13 to 0.17 mm; Size: 25 mm</td></tr><tr><td>HEPES buffer solution</td><td>ThermoFisher Scientific</td><td>15630-080</td><td>100 ml, 1 M</td></tr><tr><td>Hanks&#039; Balanced Salt Solution 1x (HBSS)</td><td>Cellgro</td><td>MT-21-022-CV</td><td>500 ml, without Calcium, Magnesium, Phenol Red</td></tr><tr><td>Papain</td><td>Worthington Biochemical Cooperation</td><td>LS003119</td><td>lyophilized powder, 100 mg</td></tr><tr><td>Magnesiumsulfate</td><td>Sigma</td><td>M-2643-500G</td><td>powder, 500 g</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>#25030-081</td><td>100 ml, 200 mM&amp;nbsp;</td></tr></tbody></table>

antibody binding specificity for kappa (v&#954;) light chain-containing human (igm) antibodies: polysialic acid (psa) attached to ncam as a case study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

此部分示出了可以通过在中枢神经系统研究人类IgM特异性的PSA抗原而获得的结果的例子。使用含有生化设置，并通过荧光免疫细胞化学具体Vκ轻链人IgM的抗体的被示出。 HIgM12从脑脑裂解物免疫沉淀的抗原，并在Western印迹用作检测试剂（初级抗体）。既不同种型对照抗体也不琼脂糖大号珠免疫沉淀的类似抗原，这表明拉下（ 图1）的特异性。使用来自WT和NCAM KO小鼠的CNS裂解物的Western印迹证明的HIgM12结合到NCAM含有的PSA（ 图2A）的特异性。使用ENDO-NF来自WT小鼠的CNS组织的酶消化标识连接到NCAM作为HIgM12该特异性结合的表位（ 图2B的PSA）。使用本文概述的方法，获得高纯度的IBA-1阳性的大鼠小胶质细胞培养物（ 图3）。市售抗PSA抗体（克隆2-2B）不标记细胞表面或内部的PSA池在固定和荧光显微镜透IBA-1-阳性神经胶质细胞。与先前公布的文献，它报告PSA-NCAM的小胶质细胞16,41无细胞表面表达HIgM12不标注在纯化的大鼠小胶质细胞表面抗原是一致的。有趣的是，固定和透化的小胶质细胞的结果在胞内，核周图案不限于特定的细胞器（ 图3）HIgM12标签。结果是在对比用抗PSA IgG抗体（克隆735）26，它在高尔基41的水平定位的PSA SynCAM在小胶质细胞获得的那些。 在对比小胶质细胞，HIgM12并在活细胞条件下（ 图4）根据高度浓缩的大鼠OPC文化A2B5靶细胞表面抗原。 OPC培养物含有〜5％的污染小神经胶质细胞。 图5示出了几乎相同的细胞表面染色HIgM12和抗PSA单克隆抗体（克隆2-2B）之间在GFAP阳性的星形胶质细胞图案（ 图5A）。 ENDO-NF-消化WT星形胶质细胞的免疫荧光标记相比使用HIgM12演示星形细胞的PSA（ 图5B）的存在下进行缓冲控制处理的WT星形胶质细胞。 体内的PSA阳性的星形胶质细胞的存在下仍有待证实。 <img alt="图1" src="/files/ftp_upload/54139/54139fig1.jpg" /> 图1:HIgM12作为"下拉"使用HIgM12从三个月大的老鼠的大脑总裂解剂免疫沉淀在免疫沉淀，人同种型控制HIgM。126或琼脂糖珠大号只为"下拉"代理商。洗脱的蛋白质在蛋白质印迹与探测针对HIgM12膜运行。从HIgM12包被的珠子洗脱蛋白的分子量为160的范围 - 200 kDa的除的IgM重链（〜65 - 73 kDa）的。这个数字已经从J.修改神经化学 15。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/54139fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54139/54139fig2.jpg" /> 图2:HIgM12目标聚唾液酸（PSA）模块上的NCAM。从P7，P13，P16和P27 NCAM A.脑组织匀浆总KO小鼠和杂同窝对照西方印迹探测与对抗HIgM12，PSA和β-actin为内参膜运行。 <strong&gt;的B点。10微克的PBS 1％NP40成人WT小鼠脑裂解液，pH值6.5的溶液用endoneuraminidase N（ENDO-N）（外）神经氨酸酶与α2-3唾液酸聚合物特异性（唾液酸酶）或PBS过夜37ºC，运行在Western印迹双峰和反对HIgM12，PSA和β-actin为内参探测。这个数字已经从J.修改神经化学 15。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/54139fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54139/54139fig3.jpg" /> 图3:粘合剂对大鼠小胶质细胞的缺失是由HIgM12标记的缺失镜像大鼠小胶质细胞培养48小时的聚-D-赖氨酸涂覆的玻璃盖玻片上，要么标记活在冰上用HIgM12或HIgM12或抗固定后。 -Psa单抗（克隆2-2B）。细胞ŧriple标有小胶质细胞标记IBA-1（绿色），HIgM12 / PSA（红色）和DAPI（蓝色）。图像显示缺乏小胶质细胞表面用HIgM12（上部行）的结合和缺乏抗PSA的（克隆2-2B）结合在大鼠原代小神经胶质细胞（下排）的细胞表面和内部的商店。根据双极形态学和不存在的IBA-1染色的，小的PSA阳性细胞（下排）中的建议是一个OPC。在固定细胞，HIgM12染色导致，这不是仅限于特定的细胞器，被认为是非特异性结合（中间行）一个punctated，核周格局。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/54139fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54139/54139fig4.jpg" /> 图4:HIgM12 瞄准细胞表面PSA-NCAM对大鼠的OPC鼠的OPC和小胶质细胞文化前进。红4天中增殖培养基上的聚-D-赖氨酸涂覆的盖玻片和三活标记在冰上用HIgM12或A2B5（绿色），内部的巨噬细胞/单核细胞标记物CD68（克隆ED-1）（红色）和DAPI（蓝色） 。图像显示细胞群体对于OPC标记A2B5（上部行）和HIgM12（下排）很少的CD68阳性的小胶质细胞（〜5％）majorly阳性。加入相衬图像DAPI揭示每个群细胞的完整性和手机号码。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/54139fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54139/54139fig5.jpg" /> 图5:HIgM12 和抗PSA单克隆抗体共标记小鼠混合胶质的星形细胞亚群。从WT和NCAM A.鼠标混合神经胶质细胞含有总星形胶质细胞，少突胶质细胞系细胞和微克KO动物LIA分别培养3天，用HIgM12（绿色），抗PSA单克隆抗体（克隆2-2B）（红色），并随后对星形细胞标志物GFAP（紫色）和DAPI（蓝色）活标记在冰上。 HIgM12和抗PSA揭示GFAP阳性星形胶质细胞WT虚拟相同的染色模式，但缺乏约束力，从NCAM KO小鼠星形胶质细胞。这个数字已经从J.修改答:根据描述，并与endoneuraminidase-NF（由玛蒂娜Muehlenhoff博士友情提供）或PBS对照处理过夜神经化学杂志 15。B.混合胶质细胞进行培养相同。细胞活标记在冰上用HIgM12（绿色），随后染色内部标记物GFAP（红色）和DAPI（蓝色）。 HIgM12目标在PBS控制细胞表面抗原处理混合胶质细胞培养而不是endoneuraminidase-NF处理混合胶质。这个数字已经从16 JNSCI修改。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/54139fig5large.jpg" target="_blank">PL</a>轻松点击此处查看该图的放大版本。 所有图像都使用相同的仪器设置在60倍和采取相同的处理。 <img alt="表格1" src="/files/ftp_upload/54139/54139table1.jpg" /> 表1:缓冲和解决方案食谱 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54139/Table1.xls">，请点击此处下载此表。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> CNS组织 </td><td> ENDO-NF / PBS </td><td> 裂解缓冲液 </td><td> 总成交量 </td></tr><tr><td> 1.鼠标WT </td><td> 67微克（0.45微升） </td><td> 2微升（12微克）ENDO-NF </td><td> 7.55微升</td><td> 10微升</td></tr><tr><td> 2. WT鼠标</td><td> 67微克（0.45微升） </td><td> 2微升PBS </td><td> 7.55微升</td><td> 10微升</td></tr><tr><td> 3. WT鼠标</td><td> 67微克（0.45微升） </td><td>没有PBS或ENDO-NF </td><td> 9.55微升</td><td> 10微升</td></tr><tr><td> 4. NCAM KO鼠标</td><td> 67微克（0.45微升） </td><td> 2微升（12微克）ENDO-NF </td><td> 7.55微升</td><td> 10微升</td></tr><tr><td> 5. NCAM KO鼠标</td><td> 67微克（0.45微升） </td><td> 2微升PBS </td><td> 7.55微升</td><td> 10微升</td></tr><tr><td> 6. NCAM KO鼠标</td><td> 67微克（0.45微升） </td><td>没有PBS或ENDO-NF </td><td> 9.55微升</td><td> 10微升</td></tr></tbody></table> 表2:P7 WT和NCAM KO小鼠中枢神经系统组织的ENDO-NF消化。

Watch this Scientific Journal Video about Antibody Binding Specificity for Kappa VÎº Light Chain-containing Human IgM Antibodies: Polysialic Acid PSA Attached to NCAM as a Case Study at JoVE.com

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

附NCAM作为一个案例研究聚唾液酸（PSA）:为河童（Vκ）抗体结合特异性含轻链人类（IgM抗体）抗体

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

antibody-binding-specificity-for-kappa-v-light-chain-containing-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.0K 次观看.  Mayo Clinic. We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (Vκ) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations. 

视频: Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

科研

癌症研究

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

生物化学

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

生物学

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

免疫与感染

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

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 ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

通过表面等离子体共振 设计 抗病毒药物

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

生物工程

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens

Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen.

附NCAM作为一个案例研究聚唾液酸（PSA）:为河童（Vκ）抗体结合特异性含轻链人类（IgM抗体）抗体

摘要

探索更多视频

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

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

通过表面等离子体共振设计抗病毒药物

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

确定放射性标记抗体与固定抗原的结合亲和力（K_D）

附NCAM作为一个案例研究聚唾液酸（PSA）:为河童（Vκ）抗体结合特异性含轻链人类（IgM抗体）抗体

摘要

探索更多视频

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

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

通过表面等离子体共振 设计 抗病毒药物

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

确定放射性标记抗体与固定抗原的结合亲和力（KD）

通过表面等离子体共振设计抗病毒药物

确定放射性标记抗体与固定抗原的结合亲和力（K_D）