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 border="0" cellpadding="0" cellspacing="0" width="1072">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:235px;">Fisher Scientific</td>
			<td style="width:119px;">MT-10-013-CVRF</td>
			<td style="width:503px;">HIGH GLUCOSE, with glutamine, with sodium pyruvate, 500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/mL, 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-2 Supplement (100X)</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>serum free, 10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum - Optima</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td>500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STERILE WATER FOR IRRIGATION</td>
			<td>Baxter Healthcare</td>
			<td>#2F7114</td>
			<td>USP-1000 ml, 12/CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7886-50MG</td>
			<td>D-Lys-(D-Lys)n-D-Lys &#183; xHBr, Molecular Weight 30,000-70,000 g/mol, 50 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin fraction V</td>
			<td>Sigma</td>
			<td>A-3294-100G</td>
			<td>heat shock fraction, protease free, pH 7,&#160; purity 98%, 100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G5767-500G</td>
			<td>C6H12O6, 
			 
			Molecular Weight: 180.16 g/mol, ACS reagent, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Recombinant Rat FGF basic Protein</td>
			<td>R&#38;D systems</td>
			<td>3339-FB-025</td>
			<td>BSA as a carrier protein, 25 ug, lyophilized, &#62;95%, 16.2 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Rat PDGF-AA Protein</td>
			<td>R&#38;D systems</td>
			<td>1055-AA-50</td>
			<td>carrier free, 50 ug, lypphilized, &#62;97%, E.coli-derived, 12.5 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Tris[hydroxymethyl]aminomethane (Tris bas</td>
			<td>Biorad</td>
			<td>#1610719</td>
			<td>1 kg, 99.8% pure, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Biorad</td>
			<td>#1610302</td>
			<td>1 kg, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP-381-5</td>
			<td>C2H5NO2, Molecular weight: 75.07 g/mol, White crystalline Powder, 5 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>NaCl, Molecular Weight: 58.44 g/mol, 5 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S0751-500G</td>
			<td>NaH2PO4, Molecular Weight: 119.98 g/mol, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Falcon 60mm 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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">75cm&#178; tissue culture flask</td>
			<td>Corning Life Sciences</td>
			<td>430720U</td>
			<td>75cm&#178; U-Shaped Canted Neck Cell Culture Flask with Plug Seal Cap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce&#8482; 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 height="21">
			<td height="21" style="height:21px;">4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>456-1094</td>
			<td>10-well, 50 &#181;l, for use with Mini-PROTEAN electrophoresis cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilon-P Membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>PVDF, 0.45 &#181;m, 26.5 cm x 3.75 m roll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Polysialic Acid-NCAM Antibody, clone</td>
			<td>Millipore</td>
			<td>MAB5324</td>
			<td>50 microliters, Host: mouse, monoclonal IgM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XT sample buffer (Western blot)</td>
			<td>Biorad</td>
			<td>#1610791</td>
			<td>10 ml, 4 x premixed protein sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Biorad</td>
			<td>#1610710</td>
			<td>25 ml, 98 % pure, 14.2 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endoneuraminidase-N (Endo-N)</td>
			<td>ABC Scientific</td>
			<td>ABC0020</td>
			<td>50 microliters, 200 &#181;g/ml, 3500 U/mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mm glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-102</td>
			<td>Circles No. 1; Thickness: 0.13 to 0.17mm; Size: 25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15630-080</td>
			<td>100 ml, 1M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; 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 height="21">
			<td height="21" style="height:21px;">Papain</td>
			<td>Worthington Biochemical Cooperation</td>
			<td>LS003119</td>
			<td>lyophilized powder, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesiumsulfate</td>
			<td>Sigma</td>
			<td>M-2643-500G</td>
			<td>powder, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td>#25030-081</td>
			<td>100 ml, 200 mM&#160;</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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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 VÃŽÂº 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

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

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

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

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

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

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

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

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间:应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...