Name: the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity
Uploaded: 2016-12-14T12:30:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity at JoVE

This study was supported by a research grant from the Ministry of Science and Technology of the Executive Yuan of Taiwan (NSC99-2320-B-002-011). I would like to acknowledge the excellent service provided by the Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory in College of Medicine of National Taiwan University.

人体外周血必须从知情同意下健康供者获得，利用血液样本必须符合个别机构审查委员会制定的指导方针批准。在这项研究中，要使用的协议人体血液流式细胞术（ 图1）和酶联免疫斑点试验（ 图3）的结果的示威是经台大医院的内部审查委员会（协议号201307019RINB）。 1.分离人外周血B细胞的分离纯化<ol><li>绘制〜10毫升的血液从肘正中静脉（在肘窝前部到肘部）到含有K 2 EDTA（1.5至2.0毫克/毫升血）有15毫升管中并立即翻转试管几次，以防止凝块形成。 </li><li>加的高压灭菌（121℃，15分钟）的红血细胞（RBC）裂解缓冲液（150mM的氯化铵 ，10毫KHCO 3，和1mM EDTA 35毫升，Pħ7.4）到含有新鲜血液样品（≥3的管:1体积/体积）并在室温（RT）下孵育不超过5分钟。 注:通过管透光的外观表示RBC裂解完成。 </li><li>离心在在RT 600 xg离心5分钟。确保粒料的颜色为白色。 </li><li>用10mL高压灭菌磷酸缓冲生理盐水的悬浮颗粒（PBS，137 mM氯化钠，2.7毫摩尔的KCl，4.3毫摩尔的 Na 2 HPO 4，和1.47毫摩尔KH 2 PO 4; pH7.4）和离心机如在步骤1.3。 </li><li>弃去上清液，用10ml的RPMI 1640培养基（补充10％胎牛血清，100U / mL青霉素/链霉素，0.25微克/毫升两性霉素B，和2mM L-谷氨酰胺）重悬沉淀，然后镀细胞分化成一个10厘米的培养皿（10毫升每皿血）。放置在37℃培养箱，5％CO 2的30分钟的盘。 </li><li>轻轻地摇晃培养皿几次，放置培养基（悬浮细胞）到15毫升的塑料锥形管。丢弃在培养皿中贴壁细胞（主要是巨噬细胞）。 </li><li>离心在在RT 600 xg离心5分钟。弃去上清液。 </li><li>悬浮用1mL的RPMI 1640培养基中的小球。计数与血球或自动细胞计数器的细胞数。 注:分离白细胞存活率通常是大于90％通过台盼蓝排除。 </li><li>离心机如在步骤1.7和5的浓度重悬在冷的PBS缓冲液（0.5％牛血清白蛋白（BSA）和2mM EDTA）中的〜200μL的细胞- 10×10 6个细胞/ mL。 </li><li>添加5μL的每10 6个细胞的特异性血细胞（B细胞的负选择）生物素化的抗-人抗体鸡尾酒的，并在冰上孵育30分钟21。 注:抗人抗体鸡尾酒至少应包括具体到CD2（或CD3），CD14，CD16和腹肌。 </li><li>添加10倍过量体积无菌PBS的细胞，离心分离，在600×g离心5分钟，并弃去上清液。 </li><li>添加链霉抗缀合的微珠等量（每10 6个细胞5微升）到颗粒并充分混合。 </li><li>在一个15毫升的塑料锥形管在冰上孵育30分钟。 </li><li>加入2毫升的PBS缓冲液到管。 </li><li>放置管进入一个磁性底座，并在室温下孵育8分钟。棕色微珠将逐渐附着在管壁的磁铁22旁边。 </li><li>与残留在磁性支架上的管中，小心地将上清液转移到一个新的无菌的15毫升管22。 </li><li>重复步骤1.14至1.16，并结合包含不变的B细胞两个上清液。丢弃微珠。 </li><li>离心在在RT 600 xg离心5分钟。弃去上清液。 </li><li>重悬在RPMI 1640培养基中的小球用于下游实验。 注意:通常情况下，1 - 5×10 5个 B细胞机智公顷纯度大于95％，从10毫升外周血可以分离23。 </li></ol> 2.分离的B细胞纯化和记忆的分离和普通B细胞<ol><li>使用从步骤1纯化的细胞，并确定使用血细胞计数器或自动细胞计数器的细胞数。 </li><li>重悬在100微升冷PBS缓冲液中的细胞在600 xg离心离心和RT 5分钟后。 </li><li>添加1 - 2每10 6个细胞的生物素化的CD27单抗的微克，并在冰上孵育30分钟。 </li><li>在600 XG添加10毫升的PBS缓冲液的管中，离心5分钟。 </li><li>弃上清，重悬细胞在PBS缓冲液50微升。 </li><li>添加链霉抗磁微珠的等量（1 - 2微克/ 10 6个细胞）的细胞在15毫升的塑料锥形管中。 </li><li>轻轻拌匀，在冰上孵育30分钟。 </li><li>添加2 - 3毫升PBS缓冲液到管。 </li><li>放置围成一个磁性底座和孵化在室温进行8分钟，以允许棕色微珠附着到最靠近磁体的一侧。 </li><li>与在磁性支架上的管中，小心地转移上清液部分到一个新的无菌试管中。该馏分包含富集CD27 -幼稚B细胞。 </li><li>添加5毫升含有微珠的管，轻轻悬浮微珠（ 即，CD27 +记忆B细胞的富集级分）24-25。 </li><li>离心在在RT 600 xg离心5分钟。重悬为下游实验的RPMI 1640介质中的颗粒。 注意:通常情况下，约30 - B细胞的60％，可以从健康供体7，25-26的PBMC中的CD27 +记忆细胞纯化。 </li></ol> 3.细胞为普通B细胞，记忆B细胞和PBS / PC的收集整理<ol><li>使用从步骤1纯化的细胞，确定使用hemocytome细胞数之三或自动细胞计数。 </li><li>重悬在冷的PBS缓冲液将细胞以10 7每毫升的5毫升聚苯乙烯管的浓度。 </li><li>添加1 - 2每10 6个细胞的人IgG微克，并在冰上孵育10分钟与Fc块。 </li><li>添加1微克的每一个抗CD19-APC（克隆:HIB19），抗CD27-eFluor450（克隆:O323），和抗CD38-PE（克隆:HIT2）每10 6个细胞;拌匀，在冰上孵育30分钟4，27。 </li><li>在步骤3.4的最后5分钟，加入商业7氨基放线菌素D（7-AAD）的5微升。 </li><li>在600 XG添加2毫升的PBS于管，涡旋，离心5分钟。 </li><li>悬浮细胞在1的浓度分拣缓冲液（无菌PBS用2％BSA和2mM EDTA） -在一个15毫升管每毫升5×10 7个细胞。 </li><li>过滤细胞通过尼龙网细胞滤网（40微米孔径），以消除细胞团块。 </li><li>分离细胞用流式细胞排序器配备了三个激光器:紫（405纳米），蓝色（488纳米），和红色（640纳米）。 注:单独的蓝色激光进行三色流式细胞27-28流量就足够了。 </li><li>排序细胞成三个15-mL管（含有5 RPMI培养基中的溶液），用于幼稚B细胞的同时采集（CD19 + CD27 - ），记忆B细胞（CD19 + CD27 +），和PBS /个人电脑（CD19 + CD27 + / HI CD38 +）3-4，28。 注:在步骤4中所述排序天真和记忆B细胞可以培养。 </li></ol> 4. 在离体人CD19 + B细胞，CD19 + CD27 +记忆B细胞和CD19 + CD27 体外分化-普通B细胞<ol><li>利用在步骤1.19，2.10，2.11和3.10纯化的细胞，确定与血球或自动细胞计数器的细胞数。 </li><li>悬浮用RPMI 1640培养基中的细胞以1:1的浓度- 10×10 5个每毫升和等分它们放入一个12孔板的孔中。 </li><li>在5微克/ 10 6细胞添加CPG（2006年ODN）/毫升18，29。 </li><li>培养的细胞在37℃培养箱，用5％ 的 CO 2 5天。 </li><li>收获来自每个细胞孔，分别将它们放入15-mL管中，加入5毫升的PBS至每个管，并在600 xg离心和RT 5分钟离心它们。 </li><li>算使用血球或自动细胞计数器细胞。悬浮细胞以1:1的浓度- 10×10 5个每毫升的RPMI 1640培养基。 </li></ol> 5.酶联免疫斑点检测<ol><li>在蒸馏水中添加35％乙醇的30微升到的ELISpot平板的每个孔中进行30秒。 注:吹打时，请避免在任何时候都接触在孔膜。 </li><li>倒置的ELISpot板以除去乙醇。 </li><li>把150蒸压DDH 2 O的的微升到每口井和孵化他们在RT 5分钟以冲洗掉残留的乙醇;按照与在RT无菌PBS洗涤3分钟。 注意:步骤5.1至5.3可以是可选的，这取决于板的制造中。 </li><li>放5微克/毫升的多克隆的F（ab&#39;）抗人Ig抗体（IgG + IgM阳性的IgA）2片段的50微升（在PBS中）插入的ELISpot平板的每个孔中，并在4℃下孵育他们过夜（优选的）或37℃2小时11。 注意:用Parafilm密封板的边缘，直到使用。 </li><li>倒置平板在RT以除去未结合ABS，添加的PBS 200微升至每个孔，并孵育他们两次，每次3分钟。 </li><li>添加的PBS200μL的含5％牛血清白蛋白（或RPMI1640培养基），以每孔用于阻挡，并在室温下孵育他们2小时。 </li><li>倒置平板以除去封闭缓冲液。洗各孔用PBS，200μL的两倍，在步骤5.5。 </li><li>加入100微升的RPMI 1640培养基中，以每个孔，并在37°C孵育它们。 </li><li>当准备种子细胞，反转板卸下RPMI 1640培养基。 </li><li>种子100微升（5×10 4），50微升（2.5×10 4），和25微升（1.25×10 4）的细胞（从步骤1.19，2.10，2.11，3.10，和4.6）转换成的一个孔中酶联免疫斑点板。用RPMI 1640培养基，使体积达到150微升/孔。 注:最小电镀细胞的一个或两个2倍连续稀释的建议。 </li><li>孵育在37℃培养箱板用5％CO 2的10 - 14小时18。避免孵育期间移动所述板。 </li><li>倒置平板以除去细胞和RPMI 1640培养基。 </li><li>加入200微升/孔的PBS-T（PBS含0.05％吐温20），并培育它们的5倍于RT，每次3分钟。反转板，除去每个5洗涤的洗涤液。 </li><li>添加任一的山羊抗人IgG-碱性磷酸酶（AP），与Fcγ特异性ABS（IgG的检测，1:5000于PBS-T的）或山羊抗人IgM-AP，Fcμ片段特异性ABS（对于IgM的检测，1:5000于PBS-T）到指定的孔，并在室温孵育在黑暗中2小时。 </li><li>洗各孔用PBS，200μL的两倍，在步骤5.5。 </li><li>添加50μL/孔的溴氯吲哚磷酸硝基蓝四唑（BCIP / NBT）底物溶液。紫色的斑点通常出现在5 - 15分钟。 </li><li>加入100微升/孔的DDH 2 O操作防止斑点的过度发展。 </li><li>与所有景点的全面发展后，自来水冲洗板。 </li><li>取出板的暗渠，并允许他们在黑暗中空气干燥。 </li><li>用自动板读数器与图像采集/分析单元（ 例如，一个自动扫描仪或通过在解剖显微镜手动）计数斑点。 注:该板可以存储在RT在黑暗中和后进行分析。 </li></ol>

The author declares no competing financial interests.

分离和人外周血B细胞的分离纯化通常情况下，红细胞可以有效地破裂并通过裂解缓冲液（步骤1.2）清零。它不孵育用RBC裂解缓冲超过5分钟长的PBMC是重要的，因为细胞生存力可能由氯化铵的影响。可替代地，RBC和血小板可以同时通过以下方案除去。 1，血液到缓冲液:在9:1的体积比;混合新鲜全血酸 - 柠檬酸盐 - 葡萄糖（ACD）缓冲液（pH 7.4 39 mM柠檬酸，75mM柠?...

B细胞发挥体液免疫的发展中发挥中心作用。他们最初开发在骨髓和进入血液流中作为幼稚B细胞，其可以迁移到淋巴组织，诸如脾，淋巴结，扁桃体，为进一步发展。在银相遇，一些幼稚B细胞迁移到淋巴滤泡，其中生发中心B细胞可以分化成记忆B细胞和plasmablasts（PBS）/浆细胞（PC）的。虽然大多数PBS / PC的出口进入血流，少数最终驻留在骨髓经受终端分化为长寿命的计算机1。 B细胞在循环是异构的，并且在稳定状态下，PBS / PC是外周血2罕见。作为谱系特异性表面标志物的可用性的结果，流式细胞术已经成为外周血B细胞亚群的鉴定和表征的常用方法。流动cytometr的扩展应用y是在加入细胞分选仪的功能，其允许分离与B细胞的个别亚群的分离具有高纯度的。基于人类循环B细胞一般分为三个主要亚群特异性表面受体在不同的发育阶段的表达:幼稚B细胞（CD19 + CD27 - CD38 - ），记忆B细胞（CD19 + CD27 + CD38 - ）和PBS /电脑（CD19 + CD27 + CD38 +）3-4（ 图1）。普通B细胞的性质还没有遇到过AGS。然而，它们可以分化成的IgM + CD27 +记忆B细胞。虽然幼稚B细胞在表达B细胞抗原受体（BCR）相关分子（ 例如，CD19，CD20和CD22），他们在他们的免疫球蛋白库5异构均匀。大多数CD27 +记忆B细胞可以分化成CD27+ / HI CD38 + PBS /电脑6。此外，记忆B细胞和PBS / PC是多克隆并表现出发育和功能的异质性4-7。流通中的PB / PC通常短暂的，不表达CD138，但那些由落户骨髓将最终分化，成为长寿。终末分化的PC表达CD138，下调其表面8 CD27分子。因为两者的PB和PC是能够分泌腹肌的，在许多情况下，他们被共同表示为携带者。相比之下，无论是普通B细胞，也没有记忆B细胞能产生相当数量的绝对9-10。放置在适当的培养条件下6，11-15时10日-然而，分离时，两者幼稚和记忆B细胞可以分化成的ASCs 3。事实上，从携带者和CD27 CD38 在体外分化有着相似的表面衍生的表达与那些直接分离FROM外周血6。此外， 在体外分化的携带者表达CD20表面的较低水平，这同样循环PBS /个人电脑6。虽然培养衍生的ASCs都是短暂的，它们能分泌腹肌，这表明它们在功能上是胜任和能够向体液免疫。 既ELISA和酶联免疫斑点是目前最常用的方法，用以获得关于体液免疫应答功能信息。 ELISA是一个96孔基于板的测定法，它经常用于测量血清抗原特异性ABS和其他分析物（ 例如，细胞因子）的效价。这是方便和可扩展性。 ELISA被设计为使用固相酶测定来检测ABS或其它物质，如血清存在下，液体样品16英寸从血清的ELISA所述读数已被广泛地用于表示身体的免疫反应。必要收购重的工具从ELISA检测adouts是酶标仪。读者可以决定最终产品通常由辣根过氧化物酶（HRP）缀合的检测ABS和其特定的底物17的反应所得的光密度（OD）。对于报告的体液免疫应答，通过ELISA测定血清抗体水平表示在身体的ASC的集体，但不是个别，性能。此外，酶联免疫吸附未能顾及由记忆B细胞，其不分泌腹肌的参与。 像酶联免疫吸附，酶联免疫斑点是用于检测和监测外周血样品17-18中免疫应答的广泛使用的方法。酶联免疫斑点是涉及夹心ELISA的技术。在它，将细胞放置到聚偏二氟乙烯（PVDF）的96孔膜支持孔微孔板用于短期培养。 ELISPOT试验类似于上微孔板和developin进行免疫印迹克，以每孔中的PVDF膜的斑点。一种自动化的ELISpot读取器系统或人工计数立体显微镜是必需的。酶联免疫斑点的检测的免疫应答的主要优点是在携带者和细胞因子分泌细胞的定量其极好的灵敏度。它分别报告它们的功能活动，体液免疫和细胞免疫。在体液免疫功能的测定中，通过ELISA和通过ELISPOT列举的ASC的数量来确定血清抗体水平通常相关，但来自这两个测定法中的数据读出有功能的影响19-20一些差异。酶联免疫斑点的主要优点是它的方法的灵敏度。血清抗体滴度通过ELISA报告的水平呈现半定量作为OD读数，表示相对抗体水平，或更定量地，作为集中读数当包括参考腹肌的适当的同种型的已知量。与此相反，酶联免疫斑点的结果是presente-d作为携带者的在感兴趣的细胞池的绝对数量（ 例如，未分级的外周血单核细胞（PBMC），并从PBMC中纯化的B细胞）。酶联免疫斑点法可以检测单个ASC，但ELISA需要来自的ASCs抗体达之前测量达到优化的测定依赖浓度。因此，酶联免疫斑点是定量的灵敏度明显优于ELISA。此外，酶联免疫斑点法也适用于量化从激活记忆B细胞的体外分化的携带者。记忆B细胞不分泌腹肌但能分化成在激活时的ASC;于是他们就用ELISA法检测血清中绝对没有任何贡献。因此，酶联免疫斑点是选择在培养活化后循环记忆B细胞的免疫应答的测定方法。它允许对维持长期体液免疫的监测。

<table><tbody><tr><td>BD Vacutainer K2E</td><td>BD Biosciences</td><td>367525</td><td>10 mL tube</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-02</td><td>endotoxin-free</td></tr><tr><td>Trypan blue 0.5% solution</td><td>Biological Industries</td><td>03-102-1B</td><td></td></tr><tr><td>IMag Human B lymphocyte enrichment set</td><td>BD Biosciences</td><td>558007</td><td></td></tr><tr><td>Biotinylated CD27 mAb</td><td>Biolegend</td><td>302804</td><td>clone O323</td></tr><tr><td>Streptavidin magnetic microbeads</td><td>BD Biosciences</td><td>9000810</td><td></td></tr><tr><td>15 mL Falcon tubes</td><td>BD Falcon</td><td>352196</td><td></td></tr><tr><td>Blue nylon mesh cell strainer, 40 &amp;mu;m</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Anti-human CD19-APC</td><td>Biolegend</td><td>302212</td><td>clone HIB19</td></tr><tr><td>Anti-human CD27-eFluor 450</td><td>eBioscience</td><td>48-0279-42</td><td>clone O323</td></tr><tr><td>Anti-human CD38-PE-Cy7</td><td>Biolegend</td><td>303516</td><td>clone HIT2&amp;nbsp;</td></tr><tr><td>Anti-human CD38-PE-Cy7</td><td>BD Biosciences</td><td>560677</td><td>clone HIT2&amp;nbsp;</td></tr><tr><td>Anti-human CD45-FITC</td><td>Biolegend</td><td>304006</td><td>clone HI30</td></tr><tr><td>Anti-human CD45-FITC</td><td>BD Biosciences</td><td>555482</td><td>clone HI30</td></tr><tr><td>Anti-mouse/rat/human CD27-PerCP Cy5.5</td><td>Biolegend</td><td>124213</td><td>clone LG.3A10</td></tr><tr><td>Anti-human CD27-PerCP Cy5.5</td><td>BD Biosciences</td><td>65429</td><td>clone L128</td></tr><tr><td>Anti-human CD19-FITC</td><td>Miltenyi Biotec</td><td>130-098-064</td><td>clone LT19</td></tr><tr><td>Anti-human CD19-FITC</td><td>GeneTex</td><td>GTX75599</td><td>clone LT19</td></tr><tr><td>Anti-human CD20-FITC</td><td>BD Biosciences</td><td>555622</td><td>clone 2H7</td></tr><tr><td>biotinylated anti-human CD27</td><td>Biolegend</td><td>302804</td><td>clone O323</td></tr><tr><td>biotinylated anti-human CD27</td><td>eBioscience</td><td>13-0279-80</td><td>clone O323</td></tr><tr><td>7-aminoactinomycin D (7-AAD)</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>CpG (ODN 2006)&amp;nbsp;</td><td>InvivoGen</td><td>tlrl-2006</td><td>type B CpG</td></tr><tr><td>Recombinant human IL-2</td><td>PeproTech</td><td>200-02</td><td></td></tr><tr><td>Recombinant human IL-10</td><td>PeproTech</td><td>200-10</td><td></td></tr><tr><td>Recombinant human IL-21</td><td>PeproTech</td><td>200-21</td><td></td></tr><tr><td>Recombinant human sCD40L</td><td>PeproTech</td><td>310-02</td><td></td></tr><tr><td>Protein A of &lt;em&gt;S. aureus &lt;/em&gt;Cowan (SAC)</td><td>Sigma-Aldrich</td><td>82526</td><td></td></tr><tr><td>Pokeweed mitogen (PWM)</td><td>Sigma-Aldrich</td><td>L9379</td><td></td></tr><tr><td>MultiScreen filter plates, 0.45 &amp;micro;m pore size</td><td>Merck Millipore</td><td>MSIPS4510</td><td>sterile, clear 96-well filter plate with hydrophobic PVDF membrane</td></tr><tr><td>BCIP/NBT solution</td><td>Sigma-Aldrich</td><td>B6404</td><td></td></tr><tr><td>BCIP/NBT single reagent, alkaline phosphatase substrate</td><td>Merck Millipore</td><td>ES006</td><td></td></tr><tr><td>Human IgG</td><td>Jackson ImmunoResearch</td><td>009-000-003</td><td></td></tr><tr><td>Human IgG, Fc fragment</td><td>Jackson ImmunoResearch</td><td>009-000-008</td><td></td></tr><tr><td>F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; fragment of goat anti-human Ig (IgG + IgM + IgA)</td><td>Jackson ImmunoResearch</td><td>109-006-127</td><td></td></tr><tr><td>Goat anti-human IgG-alkaline phosphatase, Fc&amp;gamma; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-055-008</td><td></td></tr><tr><td>Goat anti-human IgM-alkaline phosphatase, Fc&amp;micro; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-055-043</td><td></td></tr><tr><td>Goat anti-human IgG-peroxidase, Fc&amp;gamma; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-035-008</td><td></td></tr><tr><td>Goat anti-human IgM-peroxidase, Fc&amp;micro; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-035-095</td><td></td></tr><tr><td>BD ELISPOT AEC substrate kit</td><td>BD Biosciences</td><td>551951</td><td></td></tr><tr><td>C.T.L. ImmunoSpot analyzer</td><td>C.T.L.</td><td></td><td></td></tr></tbody></table>

the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity

体液免疫的特点是，以产生功能性的ASC，其合成和分泌特异性抗原（银），ABS等的病原体，并用于宿主防御。用于定量测定的个体的体液免疫应答的功能状态，既血清ABS和循环的ASC通常为功能读数测定。在人类中，外周血是可用于由主机B细胞引起的体液免疫应答的确定最方便和最容易获得的样本。不同B细胞亚群，包括的ASC，可直接从外周血通过选择与谱系特异性抗体共轭微珠或通过细胞仪具有流量分选分离。此外，纯化的幼稚和记忆B细胞可被激活并分化成在培养的ASCs。的ASCs促进抗体分泌的功能活性可以通过ELISPOT进行量化，这是收敛酶的测定法- 连接immunoabsorbance吸附试验（ELISA）和Western印迹技术，使个人的ASC的枚举在单细胞水平。在实践中，ELISPOT测定已越来越多地用于评估，因为易于大量血液样品的处理的疫苗效力。从外周血中分离的人类B细胞的方法中，B细胞的分化为携带者，和酶联免疫斑点的就业总IgM-和IgG-的ASC的定量将在这里描述。

外周血单个核细胞被耗尽的红细胞和贴壁细胞（步骤1.2到1.7）的。细胞的等分试样（2×10 6）进行流式细胞分析来说明幼稚B细胞，记忆B细胞和外周血（ 图1）PBS / PC的人口。在这个供体的外周血单个核细胞，淋巴细胞的约10％是CD19 + B细胞。在B细胞室，CD19 + CD27的百分比-幼稚B细胞是在50％左右。另一方面，所述CD19 + B细?...

Watch this Scientific Journal Video about The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity at JoVE

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

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 ...

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JoVE Journal

Immunology and Infection

15.1K 次观看.  National Taiwan University. 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.

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

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

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 ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

Source: Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed following pathogen re-exposure, thus it is critical to be able to detect these cells. Therefore, to clearly assess antigen-specific memory B-cell responses, both ELISA and ELISPOT should be used (2).
ELISPOT assay uses a plate containing membrane-lined wells that are coated with antibodies in order to capture secreted proteins of interest. Then, the plate is loaded with cells and stimuli to induce protein production. The secreted proteins are captured by the antibodies coated on the surface. After appropriate incubation time, cells are removed and the secreted molecule is detected by using a biotinylated antibody that is specific for a different epitope, as compared to the capture antibody. Next, streptavidin peroxidase is added, followed by the addition of a substrate that allows the detection of the spots (Figure 1). The strength of this assay is that it allows one to quantitate the number of cells producing the protein of interest. Importantly, one can assess if there are changes in the total number of cells producing a specific protein or if individual cells within a population are producing more protein. Moreover, it can provide information regarding kinetics and can be used to assess overall immune activation (mitogen stimulation) relative to antigen-specific responses (antigen simulation). The ELISPOT assay will permit the detection one activated cell amongst 300,000 cells following mitogenic or antigen-specific activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" /> 
Figure 1: ELISPOT protocol overview.
The major advantages of this assay are its- a. Simplicity- the protocol is relatively simple and straightforward. It does not require technical expertise, b. Sensitivity- it permits the detection of immune cells at the single cell level and requires very few cells compared to other methods such as flow cytometry, c. Functionality- it provides quantitative data regarding immune function.
This lab exercise demonstrates the ELISPOT protocol for detection of IFN-&#947; secreting-splenocytes, but as mentioned above this assay can also be used to assess antibody secretion by B cells (3).

ELISPOT Assay: Detection of IFN-&amp;#947; Secreting Splenocytes

ELISPOT 分析:检测 IFN-- 分泌性细胞

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

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Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016).
In this video, we describe a protocol for the flow cytometry-based sorting of na&#239;ve B cell, memory B cell, and plasmablast plasma cell subpopulations from human peripheral blood B cells. The different B cell subpopulations can be distinguished based on fluorophore-based antibody binding to the CD19, CD27, and CD38 receptors expressed on the B cell surfaces, followed by cell sorting.

基于流式细胞术的细胞分选，适用于不同的循环 B 细胞群

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

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Assays and Functional Profiling

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, which correlates with the B cells&#39; secretory activity.

使用 ELISpot 测定对分泌抗体的 B 细胞进行功能评估

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.