作者要感谢从NIH 3R01AI080289 - 02S1，并根据NIH / NIAID的2P30AI060354 - 07哈佛大学艾滋病研究中心学者联谊会的资金。

1。培养吞噬细胞<ol><li>文化的THP - 1细胞的RPMI 1640 10补充10％胎牛血清作为一个在T烧瓶固定悬挂。细胞密度应保持0.5x10 6 /毫升以下，以保持一致FcγR表达和检测性能水平。 </li></ol> 2。准备生物素标记的抗原<ol><li>磺酸基NHS LC生物素试剂盒，根据制造商的指示，要biotinylate利益的目标抗原的计算量。 </li><li>溶解在水中的生物素化试剂，并立即加在缓冲区不包含伯胺抗原的计算量。允许进行反应在室温下1小时，偶尔搅拌。 </li><li>删除在一个合适的截留分子量的离心浓缩装置Amicon的缓冲交换多余的，未结合的生物素保留的轻盾T抗原。样品室顶部的浓度单位，并添加PBS，使总体积达15毫升填充线。在4000 XG离心机把音量降低到约1.5毫升。两次重复此过程将删除99％的免费素的抗原，以确保最大的涂料珠。 </li></ol> 3。准备抗原饱和珠<ol><li> 100μL1毫米的荧光neutravidin珠洗净两次在1毫升0.1％PBS - BSA后在高速离心旋转。悬浮洗涤100μLPBS - BSA和分装成10管珠。 </li><li>为了确定抗原涂层饱和珠的条件，结合两方面的步骤，在不同浓度的生物素标记的抗原10洗珠悬浮UL。在4 ° C孵育过夜在一个转子的离心管。 </li></ol>注：珠饱和度，必须确定实验。这是可以实现的确定珠涂层的条件下，产量最大的吞噬功能，当珠子随后与控制单克隆抗体的调理。 <ol start="3"><li>取出1毫升PBS - BSA和高速离心洗涤，直到珠颗粒未结合的抗原。取出PBS - BSA和重复。 </li><li>在最后1毫升的PBS - BSA量的悬浮抗原包被的磁珠。珠，可存储长达一个星期，在4℃，使用前。 </li></ol> 4。准备抗体样品<ol><li>临床抗体样品可从血浆中根据制造商的说明使用瓜胶抗体纯化试剂盒纯化。纯化的IgG可以储存在4 ° C，直到准备使用。 </li></ol>注：关于处理人体标本适当的预防措施，必须始终采取了。 <ol start="2"><li>确定在A280吸光度纯化抗体的浓度，并以1毫克/毫升的PBS稀释样品。 </li><li>准备断定Ive和负的单克隆控制抗体稀释至1 mg / ml的PBS中。 </li></ol> 5。电镀实验<ol><li>重悬洗净，抗原饱和珠溶液的制备涡旋以上，并转移到每一个圆底96孔板以及10μL。必须小心，不断搅动珠悬浮，以确保数目相等的荧光珠，每孔加入。 </li><li>添加不同的利益，以每口井的抗体浓度，创造了每个抗体的剂量反应曲线。根据目前的抗体滴度样品之间的最佳浓度会有所不同，但0.01 - 100微克/毫升终浓度范围将提供良好的覆盖，并允许感兴趣的浓度范围内的鉴定。确保抗体被添加在容量不超过20μL。 </li><li>孵育珠和抗体样品为2小时37 ° C至使抗体opsonize的珠子。&lt;/ LI&gt; <li>准备在2.5 × 10 5细胞/ ml的THP - 1细胞的悬浮液，添加200μL这种悬挂，每孔共5 × 10 4 THP - 1细胞，每孔。 </li><li>在37孵育过夜℃，5％CO 2，在一个固定的孵化器，使细胞和珠通过重力颗粒。 </li></ol>注：信噪比可确定最佳珠的改进：抗体：THP - 1细胞比例，对于一个给定的抗原和抗体的来源。 6。流式细胞仪分析<ol><li>取出100μL上清液，每孔小心，不要打扰细胞沉淀。上清可以保存，如果分泌细胞因子的确定或其他分析所需。 </li><li>固定，每口井和吸管添加的4％多聚甲醛100μL重悬和混合细胞。 </li><li>可以使用一个BD LSR II配备HTS酶标仪高通量流式细胞仪分析。</li><li>程序软件组合，每孔三次（100μL混合体积）和分析30μL，每个样品或至少2000细胞活动。 </li><li> FlowJo或相同的软件，收集的数据进行分析。有用的指标包括珠+％，或荧光的细胞，它提供了一个衡量目前的吞噬细胞的数量，以及荧光强度的吞噬细胞，它提供了一个吞噬珠数量的措施。这些值相乘，产生一个综合的平均荧光强度，或iMFI。 </li><li>除以平均荧光珠1 iMFI，可以计算出平均每个吞噬细胞吞噬珠。 </li></ol> 7。代表性的成果受影响和不受影响的科目应的抗体样本有明显的差异。图1A呈现艾滋病毒抗体的样本负的流式细胞仪直方图tive（黑色曲线）和艾滋病毒阳性（灰色跟踪）的问题，并示范带动增加的吞噬抗原特异性抗体的存在。 最佳灵敏度检测的是依赖于饱和与生物素抗原的珠子。图1B提出观察时，涂有不同数量的抗原珠3单克隆抗体（包括非约束性的抗体，三角形）调理吞噬作用，并确立作为这种抗原的饱和浓度2μg的抗原/μL珠。 剂量反应曲线图2中，并演示受到抗体样本的差的能力，以诱使超过0.05-5微克/毫升的抗体浓度范围内的吞噬功能。这个差的吞噬功能，可驱动滴度或FC域的属性，如IgG亚类和糖基化状态或者差异。 当THP - 1细胞的我马吉德阿卜荧光显微镜，珠的吞噬功能有明显的证据。图3给出了两个63X抗体调理（绿色）和非调理（红色）珠孵化后的THP - 1细胞的图像，显​​示出在缺乏抗体的情况下吞噬吸收。当时间的推移显微镜，特定抗体的荧光珠的吞噬吸收更为显着（电影1，20倍放大倍率）。 以前的研究工作已经证实与细胞相关的珠子内部，原发性单核细胞的实验已同​​意与吞噬分数（iMFI值），以及在这种高通量检测（数据未显示）。 <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="图1"/> 图1。含量的质量控制。 1A，从HIV阴性的主题（黑色跟踪）和艾滋病毒呈阳性的主体（灰色跟踪）抗体样本吞噬流式细胞仪直方图。1B：实验测定的最优样本抗原珠涂层条件。抗原特异性单克隆抗体（圆形，方形）&gt; 2微克抗原/μL珠涂珠展示最大的吞噬功能，同时控制抗体（三角形）演示没有吞噬活性。 <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="图2"/> 图2。吞噬功能的剂量反应曲线。从HIV阳性（处理，未经处理的，并表现出抗逆转录病毒治疗的情况下，控制病毒复制）和HIV阴性科目gp120的驱动器的吞噬功能（艾滋病毒信封）涂珠差异的临床抗体样品。 <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="图3"/> 图3。高效的内部化。显微镜证实了抗体调理珠（绿色）的内化，而非调理珠留在溶胶ution（63X放大倍率）。 电影1。时间推移抗体驱动的吞噬功能的显微镜超过14个小时的过程中进行，并允许利用高通量的检测THP - 1细胞的吞噬活性的可视化。绿色荧光珠抗体的调理，红色荧光珠提供了一个阴性对照。 <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">点击这里观看影片。</a>

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没有利益冲突的声明。

这里所描述的检测可以利用一个长期利用研究吞噬过程10板的自动流式细胞仪的单核细胞株的吞噬活性的临床抗体样本的高通量分析。在其能够精确地刻画抗原特异性抗体的子集，这是对他人有利的检测，不仅为来自不同学科的驱动，在特定疾病的抗体细胞的吞噬功能差异的研究，也多抗原特异性内同一主题的研究。由于先天的效应细胞，包括单核细胞和其他抗原提呈细胞的抗体招聘强烈影响疾病的结果11-13，是根据 FC域14-16 Asparagine297抗体几何，IgG亚类和糖基化，生物变量，这种方法提供评价一个采取行动的关键抗体机制。此外，因为抗体的Mediated细胞的吞噬功能是利用一些病原体在感染的7,17,18过程中，这个实验认为在评估过程中抗体依赖性增强的承诺，并突出吞噬的双重性质，作为一个潜在的保护，以及潜在的有害抗体的活性。 描述该试剂盒可用于分析从临床样本的患者人群抗体，而且疫苗接种，并可能进行调整利用，而不是纯化IgG的血清。此外，细胞因子分泌到细胞的吞噬功能，可以从培养上清进行分析，并可以通过FCR -阻断抗体，使吞噬效力不仅要确定差异决定的具体FCR影响，但下游信号事件与洞察力，机制，可能有助于解决和完善这种免疫机制的理解。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> EZ - Link的磺酸基- NHS - LC -生物素微套件</td><td>热Scientfic </td><td> 21935 </td><td></td></tr><tr><td> Amicon超15离心式过滤器单元Ultracel - 50膜</td><td> Millipore公司</td><td> UFC905024 </td><td>过滤单元大小可能会有所不同，根据抗原的大小</td></tr><tr><td> FluoSpheres NeutrAvidin标记的微球，1.0微米，黄绿色荧光（五百一十五分之五百零五） </td><td> Invitrogen公司</td><td> F - 8776 </td><td>制造商生产各种颜色产品</td></tr><tr><td>瓜凝胶抗体纯化试剂盒</td><td>热</td><td> 45212 </td><td>应采取核实SDS - PAGE的AB样品的纯度。 </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

通过聘请专业吞噬细胞的Fc受体诱导抗体驱动的吞噬功能，并可以促进两个间隙以及疾病的病理。抗体的可变区的属性一直被认为关键在体内的功能，抗体，通过他们的Fc域招聘先天免疫细胞的能力已成为越来越受欢迎，作为其疗效的主要因素，无论是在重组的设置单克隆抗体治疗，以及在自然感染或疫苗接种1-3。 重要的是，尽管它作为一个常量域的命名，抗体Fc域不具有恒定 ​​功能，是强烈的IgG亚类（IgG1的- 4）和天冬酰胺糖基化297 4-6调制。因此，这种方法，研究在临床标本中的抗原特异性抗体的功能上的差异，将促进相关吞噬锅疾病状态下，容易受到感染，恶化，或临床结果抗体的差分。 此外，这种效应功能抗体的记录，以加强提供宿主细胞通过Fc受体驱动的吞噬功能 7病原体访问的感染能力，显得尤为重要。此外，还有一些证据表明，免疫复合物的吞噬吸收，可以影响免疫反应8的Th1/Th2细胞极化。 在这里，我们描述了旨在检测​​抗体诱导细胞的吞噬功能的差异，这可能是造成微分IgG亚类，聚糖结构在Asn297，以及抗原特异性抗体的免疫复合物的能力，形成一个高吞吐量的方式检测。为此，1微米的荧光珠涂有抗原，然后培养与临床的抗体样本，产生荧光抗原特异性免疫复合物。这些antiboDY -调理珠，然后培养与单核细胞表达多个FcγRs，包括抑制和激活的行​​。含量输出可以包括细胞的吞噬活性，细胞因子的分泌，和使用的FcγRs模式，并确定在一个标准化的方式，使之成为一个非常有用的系统，在此抗体依赖的效应功能解析差异，在感染和疫苗介导的保护9。

Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

Determining the Phagocytic Activity of Clinical Antibody Samples

我们目前的高通量流式细胞仪检测，以确定从临床标本中的抗原特异性抗体的吞噬活性，利用荧光抗原包被的磁珠和单核细胞表达多个Fc受体提供一个标准化的受体的使用和吞噬活性测定重现时尚任何利益抗原。

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

determining-the-phagocytic-activity-of-clinical-antibody-samples

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JoVE Journal

Immunology and Infection

23.3K 次观看.  Ragon Institute of MGH, MIT, and Harvard. 我们目前的高通量流式细胞仪检测，以确定从临床标本中的抗原特异性抗体的吞噬活性，利用荧光抗原包被的磁珠和单核细胞表达多个Fc受体提供一个标准化的受体的使用和吞噬活性测定重现时尚任何利益抗原。

视频: Determining the Phagocytic Activity of Clinical Antibody Samples

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

确定使用血凝检测血清中的反应和滴度

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...