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。
此协议中的关键步骤包括 Ag 免疫、复古眶出血和同种特异 ELISA。对于 TD ag 免疫, 应彻底悬浮 TNP-KLH/明矾混合物, 以确保注射适量的银/佐剂。如果在吸管或毛细血管管中的血液凝块在复古眼眶出血, 立即改变为新的吸管或毛细血管管。在 ELISA 中使用的缓冲器和试剂应是明确的解决方案, 不应含有沉淀, 这可能给出假阳性结果与异常 OD405值。此外, 在井中应避免气泡在所有 ELISA 步骤, 这也可能给假阳性或假阴性结果与异常 OD405值。这些偶然的错误可以通过分析重复的样本来最小化。洗涤步骤在 ELISA 去除未绑定的试剂和抗体从井。洗涤不足会导致背景噪音高, 但过度洗涤可能会导致灵敏度下降, 去除涂层抗原或绑定抗体29。该 ELISA 协议中描述的洗涤时间的数量是根据我们的经验进行优化的。
应该指出, ELISA 具有检测极限, 通常由标准曲线的线性范围来定义。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。我们建议测试样品的系列稀释, 以选择适当的稀释因子, 以计算在该协议中所描述的玻璃的浓度。但是, 如果实验结果表明, 在标准曲线的线性范围内测试的稀释因子没有给出结果, 则需要使用稀释因子根据初始 elisa 结果进行调整来进行额外的 elisa。如果所有的稀释测试低于检测限制, 原始血清样本和较小的稀释因子需要使用。如果 OD405值不显示比例下降与增加稀释因子的所有系列稀释测试, 这表明, 捕获 Ab 或涂层 Ag 是饱和的所有稀释样品和进一步更高的稀释因素需要使用。根据这项议定书, 将在2回合的 ELISA 中获得结论性的结果。
已知影响小鼠抗体应答的因素包括菌株 (遗传背景)、性别、年龄、饮食和动物设施环境30、31、32、33、34.例如, 虽然许多小鼠菌株产生 IgG2a, 但某些菌株, 如 C57BL/6 小鼠不会产生 IgG2a, 而是产生 IgG2c, 而不是35,36。最近的证据还确认共生微生物群是影响抗体应答37、38、39、40的一个因素。考虑到这些因素, 我们建议使用与性别匹配的, 年轻的成人窝的不同基因型, 共享相同的父母和笼子的 TD 和 TI Ag 免疫实验。此外, 对于同一基因型的小鼠, 经常观察老鼠对老鼠的变异 (图 4-6), 每个基因型或组需要足够的复制数 (通常, n > 8), 以获得统计有意义的结果。
同种特异 ELISA 法用于测定不同 isotypes 的滴度。然而, 单靠这种方法还不足以揭示同种滴度中观察到的差异的根本原因, 因此经常与多种互补方法结合使用。为了区分同种的差异是否是由不同数量的 b 细胞或 b 细胞的不同效率导致的, 流式细胞术41,42,43和酶链ImmunoSpot (ELISPOT)43,44,45可以使用。为分析 B 细胞生存、增殖和生发中心的形成, 可供选择的方法包括流式细胞术、脾 B 细胞体外培养和免疫组化染色, 其次为显微9、26 ,41,46,47。为阐明 B 细胞中同种交换反应的变化, 脾 B 细胞体外培养和生殖基因片段的定量 rt-pcr 研究是常用的41、43 ,48,49。为探讨亲和性成熟的原因, SHM 重链基因的体细胞超变异, 由 VDJ 区50、51、52的测序确定。为了了解记忆 b 细胞反应的差异, 通过流式细胞术对银免疫后不同记忆 b 细胞亚群的频率和数量进行分析, 利用最近确定的小鼠记忆 b 细胞标记, 包括 CD38、CD80、CD73、PD-L2、CD62L 和 CCR68,53,54,55。结合目前的协议, 这些互补方法将使研究人员能够在给定的实验环境中获得对抗体应答的全面理解。

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 border="0" cellpadding="0" cellspacing="0" style="width:577px;" width="576">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">VersaMax Tunable Microplate Reader</td>
			<td style="width:111px;">MDS Analytical Technologies</td>
			<td style="width:109px;">VERSAMAX</td>
			<td style="width:201px;">Equipment to read the plates</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">SOFTmax PRO 5.3</td>
			<td style="width:111px;">MDS Analytical Technologies</td>
			<td style="width:109px;">SOFTmax PRO 5.3</td>
			<td style="width:201px;">Software for the plate reader</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">GraphPad Prism</td>
			<td style="width:111px;">GraphPad</td>
			<td style="width:109px;">Prism</td>
			<td style="width:201px;">Software for graphing and statistics</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">TNP-AECM-polysaccharide (FICOLL)</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">F-1300-10</td>
			<td style="width:201px;">A TI Ag for immunization</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">TNP-KLH</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5060-5</td>
			<td style="width:201px;">A TD Ag for immunization</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">TNP(38)-BSA</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5050-10 (conjugation ratio: 38)</td>
			<td style="width:201px;">Coating Ag for TNP-specific ELISA</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">TNP(3)-BSA</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5050-10 (conjugation ratio: 3)</td>
			<td style="width:201px;">Coating Ag for high affinity TNP-specific Ig</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Imject Alum</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">PI-77161</td>
			<td style="width:201px;">Alum adjuvant for immunization</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Falcon Polypropylene tubes</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-959-11A</td>
			<td style="width:201px;">For incubation of TNP-KLH/alum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">BD Insulin Syringe</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-829-1B</td>
			<td style="width:201px;">For i.p. injection of mice</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Immuno 96-Well Plates, Flat-Bottom</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-245-61</td>
			<td style="width:201px;">For ELISA</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">Untreated 96-Well Microplates, Round-Bottom</td>
			<td style="width:111px;">VWR</td>
			<td style="width:109px;">82050-622</td>
			<td style="width:201px;">For serial dilutions of standards and samples</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Phosphatase substrate, 5 mg Tablets</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:109px;">S0942-200TAB</td>
			<td style="width:201px;">AP substrate</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Diethanolamine</td>
			<td style="width:111px;">VWR</td>
			<td style="width:109px;">IC15251690</td>
			<td style="width:201px;">A component of AP substrate buffer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgM</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1020-01</td>
			<td style="width:201px;">Capture Ab for mouse IgM</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG1</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1070-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG2a</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1080-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG2a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG2b</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1090-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG2b</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG3</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1100-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgA</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1040-01</td>
			<td style="width:201px;">Capture Ab for mouse IgA</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgE</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1110-01</td>
			<td style="width:201px;">Capture Ab for mouse IgE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgM</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1020-04</td>
			<td style="width:201px;">Detection Ab for mouse IgM</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG1</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1070-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG2a</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1080-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG2a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG2b</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1090-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG2b</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG3</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1100-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgA</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1040-04</td>
			<td style="width:201px;">Detection Ab for mouse IgA</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgE</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1110-04</td>
			<td style="width:201px;">Detection Ab for mouse IgE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgM standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">553472</td>
			<td style="width:201px;">TNP-specific IgM, Clone&#160; G155-228</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG1 standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">554054</td>
			<td style="width:201px;">TNP-specific IgG1, Clone&#160; 107.3</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Mouse IgG2a standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">556651</td>
			<td style="width:201px;">TNP-specific IgG2a, Clone&#160; G155-178</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG2b standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">554055</td>
			<td style="width:201px;">TNP-specific IgG2b, Clone&#160; 49.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG3 standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">553486</td>
			<td style="width:201px;">KLH-specific IgG3, Clone&#160; A112-3</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Mouse IgA standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">550924</td>
			<td style="width:201px;">Mineral oil-induced IgA, Clone&#160; MOPC-320</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgE standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">557079</td>
			<td style="width:201px;">TNP-specific IgE, Clone&#160; 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 在不同的免疫细胞子集中扮演不同的角色来调节抗体应答。为了检验这一假说, 我们用条件 TRAF3 击倒小鼠, 分别在 B 细胞、T 细胞或髓细胞中明确删除了Traf3基因, 确定了 TI 和 TD 同种的反应, 即9、10、 11。图 1描述了 TI 和 TD 研究的代表性免疫和血清收集时间表。代表 IgG1 和 IgG2b elisa 结果如图 2和图 3所示, 以说明 elisa 是如何工作的。这些包括稀释的标准和样品的板材设置 (图 2A), 一个图象在加法 AP 基体以后 (图 2B), OD405 的读的结果 (图 2C, 2D),标准稀释的值 (图 2E, 2F), 标准曲线 (图 3A, 3B), 稀释样品的值 (图 3C, 3D) 和血清 IgG1 和 IgG2b 的计算样品中的浓度 (图 3E, 3F)。图 4显示了天真小鼠血清中总的 isotypes 的代表性结果。与性别和年龄相匹配的 B-TRAF3 TRAF3-sufficient 控制小鼠相比, 我们在 B 细胞特异 TRAF3/- (littermate/) 小鼠的血清 IgM、IgG2a、IgG2b、IgG3 和 IgA 等方面表现出了统计学上的增高水平。B-TRAF3 小鼠的 hyperglobulinemia 是由于成熟的 TRAF3 细胞长期存活而导致的外周淋巴器官扩张的 B 细胞室9.图 5显示了 TNP 多糖和 TNP KLH 对小鼠进行免疫接种的 TI 和 TD 同种反应的代表性结果。这些结果显示了明显较高的 TI, TNP 特异性 IgG3 水平, 也提高 TD, TNP 特异性 IgG2b 水平在髓细胞特定 TRAF3/ (M-TRAF3/) 小鼠比在落马洲。这种增加的 TI IgG3 和 TD IgG2b 反应观察 M-TRAF3/小鼠可能是由于增加了促炎性细胞因子 IL-6 和 IL-12 的 TRAF3-/-巨噬细胞和 DCs 后免疫11。图 6显示了 TNP-KLH 免疫的小鼠主要和记忆反应的典型结果。这些结果表明, 在 T 细胞特异性 TRAF3 (T-TRAF3) 小鼠中, TD IgM 的主要反应和缺陷的 IgG1 主要和记忆反应均有所降低。TRAF3-/- CD4 t 细胞在 t细胞受体和 CD28 共同参与10中的活化, 导致 T-TRAF3 小鼠的不良反应。结合, 本文所描述的协议, 使我们能够描绘的具体作用, TRAF3 在不同的免疫细胞亚群调节 TI 和 TD 同种反应的小鼠。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57843/57843fig1.jpg"> 图 1: 典型的 TI 和 TD Ag 免疫和血清收集时间表.(A) TNP 多糖实验。(B) TNP-KLH 实验。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57843/57843fig2.jpg"> 图 2: 代表 IgG1 和 IgG2b ELISA.(A) 96 井板的安装包括空白井、7系列稀释 (1:2) 小鼠 IgG1 和 IgG2b 标准 (St01 到 St07), 1:10 只小鼠血清样本4系列稀释 (8) (S1 至 S8)。标准的浓度在每个标准井的底部给出。样品的稀释系数在样品的底部给出。(B) 增加 AP 衬底后5分钟的板材图像。板读结果 IgG1 (C) 和 IgG2b (D) 在405毫微米。小鼠 IgG1 (E) 和小鼠 IgG2b (F) 标准中不同稀释的 OD405 和浓度的值。轻质, 集中;BackCalcConc, 后计算浓度;OD, 光学密度;AvgOD, 复制的平均 OD;标准偏差;CV, 变异系数。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57843/57843fig3.jpg"> 图 3: 代表性 IgG1 和 IgG2b ELISA 数据分析.IgG1 (A) 和 IgG2b (B) 的标准曲线。合作效率 R2是 > 0.98 在两个标准曲线。箭头指示标准曲线的线性范围。OD405 和 IgG1 (C) 和 IgG2b (D) 在未知 (稀释血清样品) 中的浓度值。R, 范围;轻质集中对8例 IgG1 (E) 和小鼠 IgG2b (F) 的小鼠血清浓度的计算。不能被这种 ELISA 检测到<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57843/57843fig4.jpg"> 图 4: 单纯性小鼠血清总 isotypes 的代表性结果.血清是从性别匹配, 10-12 周大的天真的落马洲和 B-TRAF3/小鼠 (n = 10 为每个基因型; 遗传背景: 129xC57BL/6)。采用 ELISA 法测定血清总 IgM、IgG1、IgG2a、IgG2b、IgG3、IgA 和 IgE 的基底水平。对两尾数据进行了非配对t检验, 确定了统计意义。* 与 B-TRAF3 小鼠有显著差异 (p < 0.05);** 与 B-TRAF3 小鼠 (p < 0.01)有很大区别。这一数字已经从谢和al修改。9.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig4large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57843/57843fig5.jpg"> 图 5: 钛和 TD 同种对 TNP 多糖和 TNP KLH 免疫反应的代表性结果.性别匹配, 8-12 周大的落马洲和 M-TRAF3 小鼠 (遗传背景: C57BL/6) 接种了50µg 的 TI ag TNP 多糖 (顶板, n = 9 为每个基因型) 或100µg 的 TD ag TNP-KLH 混合与明矾 (底部板, n = 12 为每个基因型)。免疫后7天收集了血清。酶联免疫吸附法分析了抗 TNP IgM、IgG1、IgG2b、IgG3、IgA 和 IgE 的血清滴度。对两尾数据进行了非配对t检验, 分析了统计意义。* 与 M-TRAF3 小鼠有显著差异 (p < 0.05)。这个数字已经从 Lalani. 11.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig5large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57843/57843fig6.jpg"> 图 6: TNP-KLH 免疫的主要和记忆性反应的典型结果.性别匹配, 8-10 周大的落马洲和 T-TRAF3/小鼠 (n = 10 为每个基因型; 遗传背景: 129xC57BL/6) 接种了100µg 的 TD Ag TNP-KLH 与明矾混合在0天。在第一次免疫接种后的21天, 每只老鼠也接受了同一个 Ag 和佐剂的助推免疫。从7、7、14和28天的小鼠身上采集血清标本。用 ELISA 法测定血清样品中 TNP 特异性 IgM 和 IgG1 水平。图中描述了10对落马洲和 T-TRAF3 鼠的结果 (平均数)。对两尾数据进行了非配对t检验, 分析了统计意义。* 与 T-TRAF3 小鼠有显著差异 (p < 0.05);** 与 T-TRAF3 小鼠有显著差异 (p < 0.01);, 与 T-TRAF3 (p < 0.001)的小鼠有显著差异。这一数字已经从谢和al修改。10.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57843/57843fig6large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.3K 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

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）:TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...