Name: characterization of thymus-dependent and thymus-independent immunoglobulin isotype responses in mice using enzyme-linked immunosorbent assay
Uploaded: 2018-09-07T14:30:00.000+00:00
Duration: 6 min 15 s
Description: Watch this Scientific Journal Video about Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay at JoVE.com

这项研究得到了美国国立卫生研究院资助的 R01 CA158402 (p 谢) 和 R21 AI128264 (p 谢), 国防部赠款 W81XWH-13-1-0242 (p 谢), 一个试点奖从新泽西州癌症研究所通过批准号码 P30CA072720来自国家癌症研究所 (p 谢), 一个布希生物医学补助金 (p 谢), 一个胜利者 Stollar 奖学金 (a Lalani), 和安妮 b. 和詹姆斯 b. Leathem 奖学金 (朱)。

本议定书遵循罗格斯大学机构动物研究伦理学委员会的指导方针。所有的老鼠都是按照 NIH 的指导方针和动物保育和使用委员会批准的一项牲畜协议使用的。
1. 小鼠的制备和单纯小鼠血清的收集
<ol><li>保持所有老鼠在一个特定的无病原动物设施的免疫实验。</li>
	<li>使用与性别匹配的, 年轻的成人 (8–12周老) 淘汰赛和 littermate 控制小鼠, 共享相同的父母和笼子进行免疫研究。</li>
	<li>规划免疫和血清收集时间表, 如图 1所示。</li>
	<li>在免疫前7天 (-7 天) 收获每只老鼠的天真血清。遵循步骤4中详述的眶出血和血清准备程序。</li>
</ol>2. TNP 多糖 (TI 抗原) 和 TNP-KLH (TD 抗原) 的制备
<ol><li>彻底溶解无菌 PBS 中的抗原粉末 (pH 7.4), 使0.5 毫克/毫升的 TNP 多糖和1毫克/毫升的 TNP KLH 库存溶液。整除每一个抗原储存溶液成0.5 毫升/管的无菌离心管, 并保持整除数在-80 摄氏度长期贮存。避免多次冰冻和解冻的抗原整除数。</li>
	<li>在注射日, 根据要注射的小鼠数量计算 TNP 多糖 (50 µg/100 µL/小鼠) 或 TNP KLH (100 µg/100 µL/小鼠) 的体积。在室温下解冻适当数量的 TNP 多糖或 TNP KLH 整除数。</li>
	<li>对于 TNP-KLH 注射液, 首先稀释明矾佐剂 (40 毫克/毫升), 3 卷无菌 PBS, 最终浓度为10毫克/毫升。在稀释前要确保明矾佐剂的混合物悬浮良好。</li>
	<li>将10毫克/毫升明矾佐料浆的等量量从步骤2.3 和解冻后的 TNP-KLH 溶液 (1 毫克/毫升) 结合到5毫升聚丙烯管中, 混合良好, 并在37摄氏度孵育30分钟. 在注射前准备这 TNP-KLH/明矾混合。</li>
</ol>3. 小鼠免疫
<ol><li>采用腹腔 (ip) 注射 TNP 多糖或 TNP-KLH/明矾, 在生物安全柜中用1毫升胰岛素注射器对小鼠进行免疫。在每只老鼠的右下象限, 最好插入针在近似的30°角度, 以防止注射入内脏。</li>
	<li>在 0 天为 TI Ag 免疫注射100µL TNP 多糖每只老鼠 (图 1A)。</li>
	<li>在 0 天为 TD Ag 免疫注射200µL TNP-KLH/明矾混合 (新鲜地在步骤2.4 中准备) 每只老鼠。在21天重复同样的注射, 作为记忆研究的助推剂免疫 (图 1B)。</li>
	<li>注射后, 把每只老鼠都送到笼子里, 把所有注射的老鼠都留在没有病原体的情况下。</li>
</ol>4. 眶内出血及血清制剂
<ol><li>收获小鼠血清在不同的时间点: 为 TNP 多糖免疫, 收集小鼠血清在-7 和7天 (图 1A);对于 TNP-KLH/明矾免疫, 收集鼠血清在天-7, 7, 14 和 28 (图 1B)。</li>
	<li>对于眶内出血, 在生物安全柜25中麻醉每只小鼠与5% 异氟醚进行最小。执行踏板反射, 以确保每只老鼠足够的麻醉。</li>
	<li>一只手按住麻醉鼠的食指和拇指拉动眼球周围的皮肤, 使眼球凸出的插座25。将非血气的巴斯德吸管或毛细管在45°的角度插入眼球25下方的眼窝内角。</li>
	<li>应用温和的下行压力, 轻轻旋转吸管或管子, 进入静脉, 收集150–200µL 在吸管或管中的血液。立即将血液转移到无菌的1.5 毫升离心管, 并将鼠标返回其笼中恢复。</li>
	<li>让血液样本坐在 RT 上, 以凝聚。</li>
	<li>离心凝固的血液样品在 1.3万 x g 10 分钟在4°c。将透明血清转移到血液凝块上的新的无菌1.5 毫升离心管。</li>
	<li>重复步骤 4.6, 再一次清除残余血块, 收集清血清。</li>
	<li>将血清整除为50µL/管的无菌1.5 毫升离心管, 并将血清贮存在-80 摄氏度。</li>
	<li>在不同的时间点交替眼部出血, 使每只眼睛在整个实验中最多流血两次。在末端出血, 收集多达1毫升的血液从每只老鼠安乐死前。弄死老鼠与 5% CO2跟随了颈椎脱位。 注: 脾脏 B 细胞可用于流细胞分析和体外培养研究。我们还从脾中制备基因组 DNA, 以验证每只老鼠的基因型。</li>
</ol>5. 小鼠同种特异 ELISA 法
<ol><li>
在 ELISA 前准备缓冲区和解决方案。
	<ol>
<li>准备500毫升的耦合缓冲器 (PBS, pH 值 7.4)。</li>
		<li>准备500毫升的洗涤液 (PBS t (0.05% 吐温 20), pH 值 7.4)。</li>
		<li>准备100毫升的阻断缓冲器 (PBS 1% BSA, pH 值 7.4, 存储在4摄氏度)。</li>
		<li>准备1升的基板缓冲 (1 米二乙醇胺, ph 9.8 (97 毫升的二乙醇胺在 1 L 的 H2O, ph 调整使用 10 M HCl) 和0.5 毫米氯化镁2)。用铝箔包装瓶子, 保护基板缓冲器免受光线的侵害。存储在4摄氏度。</li>
		<li>准备100毫升的停止溶液 (3 米氢氧化钠)。</li>
	</ol>
</li>
	<li>
涂上 ELISA 板:
	<ol>
<li>对于全血清同种 ELISA, 96 井免疫板, 10 µg/毫升的同种特异性捕获多克隆山羊抗小鼠 (M, G1, G2a, G2b, G3, A, 或 E) Abs 在耦合缓冲 (PBS) 100 µL/井。</li>
		<li>对于 TNP 特异性的同种 ELISA, 96 井免疫板与10µg/毫升 TNP(38)-BSA 在 PBS 在100µL/好。</li>
		<li>为高亲和性 TNP 特异性同种 ELISA, 96 井免疫板与10µg/毫升 TNP(3)-BSA 在 PBS 在100µL/井26。一夜之间将盘子孵化4摄氏度。</li>
	</ol>
</li>
	<li>涂层孵化后, 用 PBS T 的200µL 清洗板材2次。在每次洗完后, 丢弃洗涤缓冲器和污点晾干盘子 (在一摞纸巾上翻一遍)。</li>
	<li>块板: 添加200µL/井阻塞缓冲 (PBS 中的 1% BSA) 到涂层板, 并孵化1小时在 RT 板。</li>
	<li>
当板块阻塞时, 准备稀释同种标准和血清样本, 在一个单独的, 未经处理的96井板在150µL/井的阻断缓冲。
	<ol>
<li>准备250同种标准作为起始标准浓度 (St01), 并使7至 10 1:2 串行稀释的标准 (St02 St07 或 St10,图 2A)。</li>
		<li>以1:100 或1:500 稀释因子为起始稀释剂制备小鼠血清样品, 并制作3或 4 1:10 系列稀释, 用于总的同种或1:5 系列稀释, 用于检测每种血清样品的 TNP 特异性免疫球蛋白同种 (图 2a)。对于 IgE ELISA, 以1:2 稀释因子为起始稀释剂制备小鼠血清样品, 并使每种血清样品的3为1:5 系列稀释。</li>
	</ol>
</li>
	<li>封堵后, 从步骤5.4 三次洗盘子, 200 µL/井的 PBS T 和污点干燥后, 每洗盘子。</li>
	<li>转移100µL/井稀释的同种标准 (适用于捕获和检测 Abs) 和稀释血清样品从步骤5.5 到在步骤5.6 中准备的板材。用标准和样品在一夜之间孵化出4摄氏度的板材。</li>
	<li>洗盘子3次与200µL/井的 PBS 和污点干燥后, 每洗盘子。</li>
	<li>在每个板块, 添加100µL/µg 适当的碱性磷酸酶 (AP) 共轭同种特定山羊抗小鼠的免疫球蛋白 (M, G1, G2a, G2b, G3, A, 或 E) Abs 稀释在堵塞缓冲。</li>
	<li>用 AP 共轭 Abs 在 RT 上孵化出 1-2 小时的钢板。对于 IgE 检测, 在37摄氏度的温度下孵化板材。</li>
	<li>通过将两片5毫克磷酸酶基体溶解在10毫升的基片缓冲器上, 制备1毫克/毫升的基质溶液。</li>
	<li>每次清洗后, 每盘5次, 用200µL/井和印迹干燥盘子。</li>
	<li>添加100µL/井的基板溶液到每个板块。允许反应发展在 RT, 通常只需要几分钟。</li>
	<li>
使用微板块阅读器与它的相关软件阅读每版 405 nm。
	<ol>
<li>单击 "设置" 将波长 "Lm1" 设置为 "405 nm", 然后单击 "确定"。</li>
		<li>点击 "模板", 分配 "空白" 井, "标准" 井和 "未知" 井根据96井板设置。</li>
		<li>对于 "标准" 井, 请单击 "系列", 将 "第一个样本" 设置为 "St01", 选择 "从" 开始"顶部", 并将 "复制" 设置为 "2"。检查 "示例 Descriptor" 并输入 "标准值": 选择 "单位" 作为 "ng/mL", 将 "起始值" 设置为 "250", 将 "步骤" 设置为 "2"。单击 "确定"。</li>
		<li>当最集中的标准达到1的光密度 (OD) 时, 点击 "阅读" 阅读该板块。</li>
		<li>单击 "文件" 菜单, 然后单击 "保存" 以保存文件。</li>
		<li>当最集中的标准方法分别 OD405 1.5、2和2.5 时, 单击 "读取" 以再次读取该盘。</li>
	</ol>
</li>
	<li>通过添加25µL/井的3M 氢氧化钠来停止反应。一次完成一个盘子的步骤5.12 到5.15。</li>
	<li>
使用与微板块阅读器关联的软件分析 ELISA 数据:
	<ol>
<li>检查同种标准的 OD405 值, 并选择适当的读数, 并为详细数据分析提供良好的线性范围的标准。</li>
		<li>选择4参数拟合程序以绘制标准曲线。检查标准曲线 (R2) 的协同效率, 它需要 > 0.98 才能确保良好的数据质量。</li>
		<li>检查每个样品的 OD405 值是否随着稀释因子的增加而降低。通过点击 "未知" 来检索所有稀释样品井的浓度。</li>
		<li>在同种标准曲线的线性范围内, 选取每个样品的适当井 OD405 浓度计算。</li>
		<li>用该公式计算每种样品的血清同种浓度: 血清蛋白浓度 = 选择的井浓度 x 稀释因子。</li>
	</ol>
</li>
	<li>绘制图表并使用适当的软件9、10、11进行统计分析。在同一时间点进行血清同种水平的垂直散射图进行比较。对于 TD 记忆响应的研究, 制定时间-路线反应曲线, 以检查在增压免疫前后的同种反应。使用误差线显示每个样本组的标准偏差 (SD)。为了比较两种基因型小鼠的同种反应, 对两尾数据进行配对t检验, 确定统计学意义。设置p值 < 0.05 显着不同。</li>
</ol>

作者没有竞争的金融利益。

在这里, 我们描述的协议, 以表征的 TD 和 TI 同种反应的小鼠使用 ELISA。成功实施本议定书需要使用表 1中规定的材料, 包括 ELISA 化验板、免疫 Ags、小鼠同种特异抗体和标准。应注意避免使用组织培养治疗板的 ELISA。稀释的标准和血清样品应在单独未经处理的板 (圆底), 然后添加到 ELISA 板。使用该协议, 我们一直得到可靠的结果, 具有较高的特异性和灵敏度。
此协?...

B 淋巴细胞是体液免疫的主要参与者, 是哺乳动物中唯一能够产生抗体的细胞类型, 也称为免疫球蛋白 (Ig)1,2。B 细胞分泌的抗体通过多种机制, 包括中和、opsonization 和补充活化, 为入侵病原体提供了强大的防御, 导致保护性免疫3。b 细胞分泌抗体仅在特定 B 细胞完全活化后才能实现, 通常需要两个不同的信号3。信号1由抗原 (Ag) 的直接捆绑中继到 b 细胞受体 (BCR) 在特定天真 b 细胞的表面表达3。根据信号来源 2, B 细胞活化可分为胸腺依赖性 (TD) 或胸腺独立 (TI)3,4。在 TD 抗原反应中, 信号2由活化同源 CD4 t (tH) 细胞提供, 表达 CD154, 在 B 细胞1,2,3表达的共刺激受体 CD40 的配体。在 TI 抗原反应中, 信号2来自于与收费类似的受体的接触 (TLRs 在1型 ti ag 的情况下) 或 BCRs 的广泛交联 (在2钛 ag 的情况下) 在 B 细胞3,4。1型钛 (TI-1) 抗原是 TLRs 的微生物配体, 包括细菌脂多糖 (LPS)、病毒 rna 和微生物 CpG DNA4、5。2型钛 (TI-2) 抗原具有高度重复性的结构, 并且能够通过 BCRs4,6的多重交联, 向 B 细胞传递长期持久的信号。TI-2 抗原的典型例子包括肺炎球菌多糖和抗原共轭多糖6,7。TD 和 TI 抗原均可诱发强健的抗原特异性 IgM 反应, 也可诱发同种交换抗体 (IgG、IgA 和 IgE) 的产生, 并借助抗原呈现细胞 (apc) 提供的帮助, 如树突状细胞 (DCs)1 ,2,3。此外, td 和 TI 抗原都能在 apc 的帮助下诱发记忆反应, 但 td 抗原在诱导记忆 B 细胞生成3、8时更有效。
在本议定书中, 通过腹腔 (ip) 免疫与抗原共轭模型抗原 24, 6-trinitrophenyl 锁孔帽贝血蓝蛋白 (TNP KLH) 和 TNP 多糖 (中性, 高度分枝) 诱发小鼠的 TD 和 TI 的反应。和高质量), 分别为9,10,11。TD 抗原通常与佐剂一起使用, 以提高抗体的产生12。在我们的协议中, TNP KLH 注射了明矾, 这是免疫研究中常用的佐剂12。其他可以使用的佐剂的例子包括完整或不完整的弗氏的辅助 (CFA 或 IFA), 单磷酰脂 A/海藻糖 dicorynomycolate ("Ribi" 佐剂), 和中央核苷酸等13, 14. 免疫后, 小鼠血清在不同的时间点收获, 血清中的 TNP 特异抗体使用同种特异的酶联免疫吸附试验 (ELISA)9、10进行量化,11。
ELISA 是一种以板材为基础的检测方法, 广泛应用于医学诊断工具, 也是生物医学研究15、16的分析工具。它用于检测和量化包括抗体、激素、细胞因子、趋化因子和各种抗原等的物质。elisa 可以用几种不同的格式进行, 包括直接、间接、三明治和竞争性 elisa15、16。一般而言, 它涉及将抗原固定在固体表面, 通常是一个96井微量滴定板, 它是用一个主要抗体孵化的。孵化后, 未绑定抗体被冲走。在直接 ELISA 中, 主要抗体直接与一种酶 (典型的辣根过氧化物酶或碱性磷酸酶) 结合, 它可以将显色基底切割成一个由信号检测仪器检测到的可见颜色变化, 如分光光度计15,16。相比之下, 如果用酶链二次抗体来结合原抗体, 那么这被认为是间接 ELISA15,16。直接 elisa 更快, 而间接 elisa 更敏感15,16。在三明治 ELISA 中, 这些盘子上涂有一种 "捕获" 抗体, 用来固定样本中感兴趣的抗原, 然后用另一种 "检测" 抗体直接或间接地检测到捕获的抗原15,16. 三明治 ELISA 具有很高的特异性, 因为抗原是由两种不同的抗原抗体检测出来的。在竞争性 ELISA 中, 在样本抗原和板块绑定抗原之间建立了与原抗体结合的竞争, 然后通过测量基片中信号的减少来量化样本中的抗原浓度。15,16. 竞争性 ELISA 可使用上述直接或间接格式进行, 对仅有一个表位15、16的小抗原的检测非常有用。
抗体测量的替代技术包括无线电免疫分析 (RIA), 电化学发光 (ECL) 检测和表面等离子共振 (SPR) 检测17。RIA 是第一个利用核素试剂18,19测量抗原 (或抗体) 具有高度特异性和灵敏度的免疫分析法。然而, 由于对放射性毒性、处置成本、保质期以及使用放射性材料的特殊许可证等问题的关注, ELISA 是一种比较方便的常用20、21技术。ECL 是一种高度敏感的检测方法, 其中化学发光反应是利用电产生高活性物种从电极表面的稳定前体, 并可用于测量的数量的分析 (如抗原或抗体)22。但是, ECL 需要一种特殊的仪器, 因此不像 ELISA23那样广泛使用。SPR 是一种直接的检测方法, 可用于测量传感器芯片表面上固定化分子 (e.、抗原) 的配体 (e.、抗体) 的结合情况24。SPR 非常明确地检测了交互作用, 不需要使用标记试剂作为 ELISA。然而, SPR 还需要一个特殊的设备, 灵敏度低于 ELISA17。鉴于替代方法的局限性, ELISA 是本协议中最适合我们使用的技术。在这里, 我们描述了使用三明治 elisa 分析总的同种水平和间接 ELISA 的程序, 以分析抗原特异性的 isotypes。

<table><tbody><tr><td>VersaMax Tunable Microplate Reader</td><td>MDS Analytical Technologies</td><td>VERSAMAX</td><td>Equipment to read the plates</td></tr><tr><td>SOFTmax PRO 5.3</td><td>MDS Analytical Technologies</td><td>SOFTmax PRO 5.3</td><td>Software for the plate reader</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>Prism</td><td>Software for graphing and statistics</td></tr><tr><td>TNP-AECM-polysaccharide (FICOLL)</td><td>Biosearch Technologies</td><td>F-1300-10</td><td>A TI Ag for immunization</td></tr><tr><td>TNP-KLH</td><td>Biosearch Technologies</td><td>T-5060-5</td><td>A TD Ag for immunization</td></tr><tr><td>TNP(38)-BSA</td><td>Biosearch Technologies</td><td>T-5050-10 (conjugation ratio: 38)</td><td>Coating Ag for TNP-specific ELISA</td></tr><tr><td>TNP(3)-BSA</td><td>Biosearch Technologies</td><td>T-5050-10 (conjugation ratio: 3)</td><td>Coating Ag for high affinity TNP-specific Ig</td></tr><tr><td>Imject Alum</td><td>Fisher Scientific&amp;nbsp;</td><td>PI-77161</td><td>Alum adjuvant for immunization</td></tr><tr><td>Falcon Polypropylene tubes</td><td>Fisher Scientific&amp;nbsp;</td><td>14-959-11A</td><td>For incubation of TNP-KLH/alum</td></tr><tr><td>BD Insulin Syringe</td><td>Fisher Scientific&amp;nbsp;</td><td>14-829-1B</td><td>For i.p. injection of mice</td></tr><tr><td>Immuno 96-Well Plates, Flat-Bottom</td><td>Fisher Scientific&amp;nbsp;</td><td>14-245-61</td><td>For ELISA</td></tr><tr><td>Untreated 96-Well Microplates, Round-Bottom</td><td>VWR</td><td>82050-622</td><td>For serial dilutions of standards and samples</td></tr><tr><td>Phosphatase substrate, 5 mg Tablets</td><td>Sigma</td><td>S0942-200TAB</td><td>AP substrate</td></tr><tr><td>Diethanolamine</td><td>VWR</td><td>IC15251690</td><td>A component of AP substrate buffer</td></tr><tr><td>Goat anti-mouse IgM</td><td>SouthernBiotech</td><td>1020-01</td><td>Capture Ab for mouse IgM</td></tr><tr><td>Goat anti-mouse IgG1</td><td>SouthernBiotech</td><td>1070-01</td><td>Capture Ab for mouse IgG1</td></tr><tr><td>Goat anti-mouse IgG2a</td><td>SouthernBiotech</td><td>1080-01</td><td>Capture Ab for mouse IgG2a</td></tr><tr><td>Goat anti-mouse IgG2b</td><td>SouthernBiotech</td><td>1090-01</td><td>Capture Ab for mouse IgG2b</td></tr><tr><td>Goat anti-mouse IgG3</td><td>SouthernBiotech</td><td>1100-01</td><td>Capture Ab for mouse IgG3</td></tr><tr><td>Goat anti-mouse IgA</td><td>SouthernBiotech</td><td>1040-01</td><td>Capture Ab for mouse IgA</td></tr><tr><td>Goat anti-mouse IgE</td><td>SouthernBiotech</td><td>1110-01</td><td>Capture Ab for mouse IgE</td></tr><tr><td>AP-Goat anti-mouse IgM</td><td>SouthernBiotech</td><td>1020-04</td><td>Detection Ab for mouse IgM</td></tr><tr><td>AP-Goat anti-mouse IgG1</td><td>SouthernBiotech</td><td>1070-04</td><td>Detection Ab for mouse IgG1</td></tr><tr><td>AP-Goat anti-mouse IgG2a</td><td>SouthernBiotech</td><td>1080-04</td><td>Detection Ab for mouse IgG2a</td></tr><tr><td>AP-Goat anti-mouse IgG2b</td><td>SouthernBiotech</td><td>1090-04</td><td>Detection Ab for mouse IgG2b</td></tr><tr><td>AP-Goat anti-mouse IgG3</td><td>SouthernBiotech</td><td>1100-04</td><td>Detection Ab for mouse IgG3</td></tr><tr><td>AP-Goat anti-mouse IgA</td><td>SouthernBiotech</td><td>1040-04</td><td>Detection Ab for mouse IgA</td></tr><tr><td>AP-Goat anti-mouse IgE</td><td>SouthernBiotech</td><td>1110-04</td><td>Detection Ab for mouse IgE</td></tr><tr><td>Mouse IgM standard</td><td>BD Biosciences</td><td>553472</td><td>TNP-specific IgM, Clone&amp;nbsp; G155-228</td></tr><tr><td>Mouse IgG1 standard</td><td>BD Biosciences</td><td>554054</td><td>TNP-specific IgG1, Clone&amp;nbsp; 107.3</td></tr><tr><td>Mouse IgG2a standard</td><td>BD Biosciences</td><td>556651</td><td>TNP-specific IgG2a, Clone&amp;nbsp; G155-178</td></tr><tr><td>Mouse IgG2b standard</td><td>BD Biosciences</td><td>554055</td><td>TNP-specific IgG2b, Clone&amp;nbsp; 49.2</td></tr><tr><td>Mouse IgG3 standard</td><td>BD Biosciences</td><td>553486</td><td>KLH-specific IgG3, Clone&amp;nbsp; A112-3</td></tr><tr><td>Mouse IgA standard</td><td>BD Biosciences</td><td>550924</td><td>Mineral oil-induced IgA, Clone&amp;nbsp; MOPC-320</td></tr><tr><td>Mouse IgE standard</td><td>BD Biosciences</td><td>557079</td><td>TNP-specific IgE, Clone&amp;nbsp; C38-2</td></tr></tbody></table>

characterization of thymus-dependent and thymus-independent immunoglobulin isotype responses in mice using enzyme-linked immunosorbent assay

抗体, 也称为免疫球蛋白 (Ig), 分泌的分化 B 淋巴细胞, plasmablasts/血浆细胞, 在体液免疫提供了强大的防御入侵病原体通过不同的机制。疫苗接种的一个主要目标是诱导保护性抗原特异抗体, 以防止危及生命的感染。胸腺依赖 (TD) 和胸腺独立 (TI) 抗原可以引起强健的抗原特异性 IgM 反应, 也可以诱导生产同种交换抗体 (IgG, IgA 和 IgE), 以及产生的记忆 B 细胞的帮助由抗原呈现细胞 (apc) 提供。在这里, 我们描述了一个协议, 以表征 TD 和 TI 同种反应的小鼠使用酶联免疫吸附试验 (ELISA)。在本议定书中, 抗原-共轭模型抗原 TNP-KLH (明矾) 和 TNP 多糖 (PBS) 对小鼠进行了腹腔 (ip) 免疫, 从而诱发了 TD 和 TI 的反应。为诱发 TD 记忆反应, TNP-KLH 在明矾中的助推免疫接种是在第一次免疫后3周使用相同的抗原/佐剂。小鼠血清在免疫前后的不同时间点收获。血清总免疫球蛋白水平和 TNP 特异抗体随后分别使用同种特异性三明治和间接 ELISA 进行量化。为了正确量化每个同种的血清浓度, 样品需要适当稀释, 以适应标准曲线的线性范围。使用该协议, 我们一直得到可靠的结果, 具有较高的特异性和灵敏度。当与其他补充方法, 如流式细胞术,在体外培养脾脏 B 细胞和免疫组化染色 (IHC), 这项协议将允许研究人员获得全面了解抗体给定实验设置中的响应。

我们使用这个协议来调查免疫系统的一个关键调节器, TRAF3, 在 TI 和 TD 同种反应9,10,11的作用。TRAF3 直接或间接地调节了一些先天和适应性免疫受体的信号传导, 包括 TNF 受体超家族、类似收费受体和 T 细胞 receptor/CD28, 其中27,28。我们推测 TRAF3 在不同的免疫细?...

Watch this Scientific Journal Video about Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay at JoVE.com

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

本文描述了一种用 ELISA 方法表征小鼠 t 依赖性和 t 无关免疫球蛋白 (同种) 反应的协议。这种方法单独使用或结合流式细胞术将允许研究人员确定在 B 细胞介导的小鼠同种反应在 t 依赖性和 t 独立抗原免疫的差异。

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

characterization-thymus-dependent-thymus-independent-immunoglobulin

Research

JoVE Journal

Immunology and Infection

9.4K 次观看.  Rutgers University. 本文描述了一种用 ELISA 方法表征小鼠 t 依赖性和 t 无关免疫球蛋白 (同种) 反应的协议。这种方法单独使用或结合流式细胞术将允许研究人员确定在 B 细胞介导的小鼠同种反应在 t 依赖性和 t 独立抗原免疫的差异。

视频: Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

免疫与感染

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

小鼠自发性自身免疫性甲状腺炎模型的产生

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

摘要

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此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

隔离和幼稚的CD4 T细胞的Th17细胞分化

检测真正的IgE的表达小鼠B系细胞

鼠标朴素CD4

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

近一代测序鉴定小鼠与人体抗体的分泌

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

小鼠B细胞发育的流式细胞学表征

小鼠自发性自身免疫性甲状腺炎模型的产生

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

摘要

探索更多视频

此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

隔离和幼稚的CD4 T细胞的Th17细胞分化

检测真正的IgE的表达小鼠B​​系细胞

鼠标朴素CD4

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

近一代测序鉴定小鼠与人体抗体的分泌

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

小鼠B细胞发育的流式细胞学表征

小鼠自发性自身免疫性甲状腺炎模型的产生

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

检测真正的IgE的表达小鼠B系细胞