范德比尔特CTSA的成员授予1 UL1 RR024975的一部分，由国家研究资源，国立卫生研究院中心的支持。这项工作是由美国国立卫生研究院R01 HL 83839; R01人工智能65744。

对于下面的协议，我们用外周血单个核细胞（PBMC）的细胞的利息。然而，该协议可用于其他类型的细胞。使用无菌技术在组织培养罩进行检测。 第1天：板和细胞的制备<ol><li>取出ELISPOT板，并在引擎盖打开它。 </li><li> 150μL，使用多通道移液器的1X PBS洗板3次，如果有的话，是可以接受的，否则单以及吹打。 </li><li>拿出一包水库，打开引擎盖。 </li><li>新增2微克/毫升的捕获抗体，抗人干扰素-γ的，到11的1X PBS MLS。涡很好。将抗体混合水库。 </li><li>放置到每孔使用多声道100μL的涂层解决方案。 </li><li>包装封口膜板。 </li><li>板放置在冰箱过夜。该板是好几个星期。经验法则是，如果仍然在井液左，板可以用。 </li></ol>细胞制备如果是新鲜的外周血细胞计数和孵育过夜在37℃，5％的CO 2彗星。外周血单个核细胞储存在液氮，解冻的细胞是必要的。协议如下： <ol><li>在37℃水浴，直到几乎完全解冻解冻细胞的离心管。 </li><li>立即悬浮在5毫升R10的媒体。 </li><li> 5分钟旋转225 × G. </li><li>再次重悬在5毫升R10的媒体，并使用台盼蓝活细胞计数。 </li><li>将浓度2 × 10 6细胞/毫升R10的媒体。 </li><li>休息37通宵℃，5％的CO 2。 </li></ol>第2天：设立的板块<ol><li>板盖标本识别号码，以及条件和日期的标签。 </li><li>无菌1X PBS 150μL转储和blot方法，洗板6次。小心不飞溅板，同时倾销。这可能会导致板井变成紫色。 </li><li>每孔加入100μLR20的。 </li><li>孵育板在37 ° C为1小时。 </li><li>虽然该板块孵化，计数细胞休息过夜。 </li><li> 5分钟1500转的旋转细胞悬液。弃上清，悬浮颗粒，让您有一个终浓度为1X10 6每1ml细胞在R10媒体。 </li><li> 1小时的潜伏期后，取出R20的使用dump和blot方法。小心不飞溅板，同时倾销。 </li><li>加入100μL细胞液（10 5个细胞），以每口井。添加适当的肽类和/或抗原重复相应的井。使用新的枪头，每次你进入肽的工作方案。正常肽浓度为10-40微克/毫升。由于肽的不同，每个研究者应滴定肽，以获得最佳的效果。 </li><li>在阴性对照井，只有细胞（不肽添加） </li><li>在阳性对照井，添加植物血凝素（PHA 10微克/毫升; Sigma公司）。 </li><li>在37℃过夜，5％的CO 2（〜18小时） 。 </li></ol>第3天：开发板<ol><li>洗板6次，150μL，1xPBS使用dump和印迹法。 </li><li>添加100μL，PBS以及每板使用多通道移液器。 </li><li>板放入冰箱15分钟。 </li><li>取出冰箱板，并放置在组织培养罩。 </li><li>准备生物素抗体解决方案<ul><li> 0.5微克/毫升的生物素抗体（MAB 7 - B6 - 1）加入11mL PBS。 </li><li>涡的解决方案，拌匀。 </li><li>如果规划添加使用多通道的解决方案，倒入水库。 </li></ul></li><li>由弹进废纸篓，丢弃的PBS。要小心不飞溅板，同时倾销。 </li><li>将每孔100μL的解决方案。 </li><li>孵育一小时，在室温下的组织培养罩板。 </li><li> 150μL1xPBS使用转储和blot方法清洗板的6倍。在第 6 次清洗，离开板的PBS，直到你有准备的链霉亲和抗体。 </li><li>准备的链霉亲和抗体。 <ul><li>添加5.5μ升链霉亲和抗体（链霉亲和ALP）11mL PBS（1:2000稀释）。 </li><li>涡的解决方案，拌匀。 </li><li>如果规划添加使用多通道的解决方案，倒入水库。 </li></ul></li><li>留在井的倒掉PBS和杂交板。 </li><li>加入100μL链霉亲和解决方案，以每口井。 </li><li>孵育1小时在室温下的组织培养罩板。 </li><li>洗净板用PBS使用dump和杂交方法的6倍。在6 日洗，离开盘上的PBS，直到你有颜色的解决方案。 </li><li>在黑暗中工作。准备碱性磷酸酶底物溶液（碱性磷酸酶底物试剂盒四BCIP / NBT）： <ul><li>添加4滴基板试剂1至11 mL的Tris缓冲液，涡旋以及。 Tris缓冲液，涡旋以及添加4滴试剂2基板。 4底物试剂3滴添加Tris缓冲液，涡旋以及。 </li></ul></li><li>倒掉剩余的PBS和杂交板。 </li><li>加入100μL的色彩解决方案以及每板使用多声道。 </li><li>打开灯光后5至10分钟的彩色发展。 </li><li>允许色剂，以保持板，直到点开始变成紫色，很暗。这要花费5至20分钟。要小心不要离开太久基板的解决方案，因为这样做可能会引起高背景。 </li><li>用自来水清洗ELISPOT板的3倍。 </li><li>保持干燥约一个小时或隔夜。 </li><li>手动或酶联免疫斑点法酶标仪读板。 </li><li>商店板远离光线照射在一个盒子里。 </li></ol>代表性的成果： 酶联免疫斑点法的目的是对微生物抗原定量免疫反应。每个点表示一个单一的响应细胞，表达细胞因子的利益。这些点可以算作手动或使用酶联免疫斑点法酶标仪。 评估，酶联免疫斑点法已被正确地执行，应首先调查背景感兴趣的细胞因子的生产阴性对照。阴性对照井人会希望看到无斑点，虽然可能会出现一个小的背景（紫色）。通常情况下，应小于平均每口井五点。阳性对照井确认已执行的反应正确，细胞抗原刺激的反应。阳性对照井，你希望看到深紫色的背景下，由于一个点（图1）合流。应该有一点没有白色背景。如果阳性对照，阴性对照样品是合适的，可以再算上含有抗原利益的井。一个“不理想”的结果将反映高背景（井紫色），无斑点或极少数斑点，空白中心，定义不清的斑点或汇合点。提供故障排除提示列表（见表1）。 <img alt="图1" src="/files/ftp_upload/2221/2221fig1.jpg" /> 图1。ELISPOT板的代表。每个板块应包含阳性对照，阴性对照和肽在两口井最低进行的利益。 C9，C10，D9，D10的阳性对照井，你可以看到有深紫色的颜色，融合细胞和几乎没有任何白色背景。阴性对照井G11，G12，H11和H12。这些水井都没有如预期般的紫色背景和无斑点。点七国集团，八国集团，H7和H8，说明微生物肽的细胞反应;紫色斑点代表一个单一的反应细胞表达细胞因子的利益。 表1 <table border="1"><tbody><tr><td>观察</td><td>可能出现的问题</td><td>可能的解决方案</td></tr><tr><td>高背景</td><td> 1。膜两侧冲不正确 2。过多的脑细胞分泌细胞因子 3。板不妥善干 4。在开发板</td><td> 1。之前和之后的颜色发展双方的膜用蒸馏水洗净。试剂通过细胞膜进入基地的板块可能会泄漏，如果没有冲走，这些可能会导致高背景。 2。每孔细胞数量减少，这将需要优化 3。干板前阅读 4。减少开发时间</td></tr><tr><td>无斑点/很少有斑点</td><td> 1。没有足够的细胞分泌细胞因子/蛋白 2。确保正确的细胞受到刺激 3。不为l细胞王景荣不够或可能需要一段时间，到兴奋剂的反应 4。发展不足的颜色 5。没有足够的小学或中学的抗体</td><td> 1。增加细胞的数量。这将需要优化 2。还可以使用积极的刺激控制 - 一种兴奋剂，你知道会诱发细胞因子/蛋白表达 3。增加细胞孵育时间或使用间接法（预先处理的细胞与兴奋剂） 4。监测架空镜下颜色的发展，并确保发展中国家试剂已正确储存，并没有失去活性 5。将需要增加小学和/或继发性抗体浓度。这将需要优化。 </td></tr><tr><td>空白区</td><td> 1。膜没有预先处理 2。膜已经干涸了，在一些阶段 3。细胞分布不均</td><td> 1。确保膜有足够的预处理用70％乙醇（所有膜不需要这样检查与您的供应商）。用PBS 3X洗好之后 2。确保膜不会干 3。确保你混合细胞轻轻吹打入井前有一个良好的同源细胞悬液</td></tr><tr><td>空白中心</td><td> 1。从洗衣机损坏</td><td> 1。自动洗板机（或移液）流率可能过高。需要一个更柔和的洗涤程序</td></tr><tr><td>误报</td><td> 1。二级抗体聚合 2。细胞膜上的细胞仍 3。细胞培养污染</td><td> 1。过滤器的二次抗体 2。确保所有的细胞用PBS吐温20的二抗孵育前膜洗净。左膜细胞会给不规则形斑点 3。保持试剂，消毒和尽可能干净。确保您的细胞培养技术是无菌的。通过运行媒体阴性对照检查误报。板块运动， <a href="http://www.abcam.com/index.html?pageconfig=resource&amp;rid=11457#7">定义不清的</a>点</td></tr><tr><td>聚合景点</td><td> 1。标准普尔涂料，过多的抗体 2。长时间细胞培养 3。细胞过度刺激</td><td> 1。减少的主要抗体浓度 2。时间越长，细胞培养，细胞因子/蛋白，他们将分泌。这将导致更大的点，将开始合并，成为难以区别。 3。降低细胞培养步骤孵化时间。过度刺激会造成大量的细胞分泌的细胞因子/蛋白质。这会产生斑点，将启动合并，成为难以区分。在文化传媒或文化兴奋剂金额减少较短的时间</td></tr><tr><td>不好定义的景点</td><td> 1。膜不预先处理（如果你的膜需要，请与供应商） 2。细胞孵化期间的板块运动</td><td> 1。膜必须进行预处理与乙醇或这可能会导致模糊，定义不清的斑点。这将是为读者很难区分这些 2。不要让盘在细胞孵化提出的细胞会创建一个点以上。如果可能的话使用一个专门的孵化器，将不会在潜伏期打开。不要挖掘后，加入细胞板</td></tr><tr><td>阳性对照孔低</td><td> 1。不发达 - 可能是一个尚未带来室温使用链霉亲和ALP和/或BCIP / NBT解决方案的结果</td><td> 1。增加井前将试剂置于室温</td></tr><tr><td>点的密度，使得它难以量化</td><td> 1。过多的脑细胞被添加到井</td><td> 1。对细胞的稀释（即1 × 10 6，5 × 10 5，1 × 10 5，5 × 10 4，1 × 10 4细胞，每孔），以确定最佳的细胞数量，这将导致形成踩点</td></tr></tbody></table>

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酶联免疫斑点法检测已成为最重要和最广泛使用的检测，监测人体的免疫反应和各种其他物种。酶联免疫斑点法检测，免疫细胞的频率可以测量在单细胞水平上没有膨胀或操纵细胞群。酶联免疫斑点法检测已被广泛应用于各种疾病，包括感染，癌症，过敏症和自身免疫性疾病的抗原特异性免疫反应调查。这个实验中的应用已基本适用于量化是非常重要的抗原特异性细胞或特异性抗原的免疫反应，?...

A.媒体的制备<img src="/files/ftp_upload/2221/2221materialsA.jpg" alt="材料A" /> B. Tris缓冲液的制备<ol><li> 1升烧杯中加入800毫升的DDH 2 O 。 </li><li>测量的Tris 12克，并添加到烧杯中。 </li><li>放入烧杯中，加入0.12克的六水氯化镁 （MgCl 2的H 2 O ）。 </li><li>自动搅拌板带磁性搅拌棒搅拌均匀。 </li><li>一旦溶解，pH值至9.5的解决方案。 </li><li> DDH 2 O的200毫升烧杯中，使之达到至1000ml 。 </li><li> 0.22微米过滤器的过滤解决方案。 </li></ol> C.材料ELISPOT分析必要的： <img src="/files/ftp_upload/2221/2221materialsb.jpg" alt="材料B" />

enzyme-linked immunospot assay (elispot):  quantification of th-1 cellular immune responses against microbial antigens

适应性免疫是清除细胞内病原体的一个重要组成部分。检测和量化这些反应在人类的能力是一个重要的诊断工具。酶联免疫斑点法（ELISPOT）获得其对微生物的抗原，包括人群，如那些与艾滋病毒感染，移植免疫抑制，并使用类固醇的细胞免疫反应的能力，以确定的普及。此实验的能力量化针对特定微生物抗原的免疫反应，以及区分这些对策Th1或Th2型性格。酶联免疫斑点法不限于炎症部位。它是多才多艺的，在其评估的外周血，以及支气管肺泡灌洗，脑脊髓液，腹水，如积极参与网站内的免疫反应的能力。对一个或多个抗原免疫反应的检测是可能的，以及微生物蛋白质内的特定抗原决定簇。此实验有利于检测的免疫反应，随着时间的推移，以及在宿主T细胞识别抗原的区别。双色酶联免疫斑点法检测可用​​于检测两种细胞因子的同时的表达。该技术的最新应用，包括诊断为肺外结核，以及调查传染病抗原的自身免疫性疾病的贡献。

Watch this Scientific Journal Video about Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens at JoVE.com

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

可以通过使用的酶联免疫检测鉴定的微生物指标在特发性疾病的适应性免疫。

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 ...

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

34.6K 次观看.  Vanderbilt University School of Medicine. 可以通过使用的酶联免疫检测鉴定的微生物指标在特发性疾病的适应性免疫。

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

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-&amp;#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 ...

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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.

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

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 ...

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.

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

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 ...

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

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

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 ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...