1。生长的H. Capsulatum <ol><li>与真菌的工作准备了一个生物安全内阁，内阁由20分钟的紫外线照射10％的漂白粉清洗。H.在酵母阶段的 capsulatum 需要生物安全水平（BSL）二做法，而模具的形式要求BSLIII。使用酵母的形式，在下面的步骤。 </li><li>下一步准备用5毫升的PBS 15 ml锥形管。收获了殖民地的H. capsulatum（株HC ATCC G217B）从一个BHI血琼脂平板[葡萄糖10 g / L的半胱氨酸0.1 g / L的，青霉素，链霉素1％绵羊红细胞50毫升/升（科罗拉多州的血清有限公司，科罗拉多州，美国） ]耕地在37 ° C，或采取分装于-80 ° C冷冻酵母股票，并把它添加到的PBS。涡大力细胞和离心机在室温为10分钟为1100 XG。小心取出上清液互不干扰颗粒，并添加10毫升的新鲜PBS。重复这个过程三次。暂停PBS和转院至50 mL锥形管5ML细胞。 </li><li>扰乱聚合的细胞，酵母细胞悬液通过一个26 G1 / 2针ü唱10ml注射器在生物安全柜（使用面部防护）的5倍。为了隔离小，大小均匀的酵母，离心55在室温下1分钟XG的细胞，然后取出1毫升。 </li><li >细胞悬液加入到含有100毫升火腿F12培养基（GIBCO），辅以16克/ L葡萄糖，1g / L的谷氨酸，8.4mg / L的胱氨酸，6克/ L的谷氨酸，然后生长的细胞在37的无菌锥形瓶中° C为48 h，用孵化器150RPM晃动。 </li></ol> 2。单克隆抗体的杂交瘤细胞的生长和纯化<ol><li>将选定的杂交瘤细胞DMEM培养基含10％小牛血清，10％NCTC，1％非必需氨基酸和1％的青霉素，链霉素。生长的细胞在细胞培养瓶（流式细胞迪肯森）为5-7天，在37 ° C / 5％的CO 2 。 </li><li>然后离心1100 XG介质，在室温为10分钟和扰乱颗粒的情况下收集上清。 </li><li>接下来，净化的无细胞上清液使用蛋白A / G FPLC树脂。 </li><li>单克隆抗体的浓度，然后可以计算一个捕获ELISA法（1）使用一个作为标准的IgG亚型控制（图1B）。 </li></ol> 3， 组织胞浆菌 接种准备 <ol><li>以一个48小时的组织胞浆菌（ 株 HC ATCC G217B）酵母阶段文化火腿 ​​架F - 12培养基中生长。 </li><li>离心10分钟在1100 XG的细胞。弃去上清，加入新鲜的PBS。 </li><li>重复3.2程序的3倍。 </li><li>通过酵母细胞悬液通过26G1 / 2使用注射器针头的5倍。 </li><li>离心1分钟颗粒残留细胞团在55 XG暂停。上清液中含有的单细胞悬液转移到一个新的管。 </li><li>枚举单细胞悬液，用一个hematocytometer。 </li><li>调整细胞浓度，以达到1.25 × 10 7酵母（生存）或5.0 × 10 6（CFU，细胞因子和组织学）&lt;50μL（图2）suspention。 </li></ol> 4。腹腔注射的单克隆抗体<ol><li>拿起鼠标，它的尾巴和颈背（图3A）。固定鼠标，它的脖子颈背接近尽可能theears（确保采取足够的皮肤，使鼠标cannotturn其头部咬经办人;图3B和3C）。 </li><li>稳定的尾巴withthe小指轻轻对手的手掌（图3D）压。 </li><li>擦拭前放置针吸出的血液，注射前用70％乙醇注射区。 </li><li>使用26G1 / 2 piercethe注入单克隆抗体溶液中含有500μg（PBS，同型对照抗体或单克隆抗体进行测试，在1 mL PBS稀释），到thelower左或右下腹部象限（腹腔腔），与皮肤和abdominalmuscles动物头向下的位置，注意避免的隔膜和其它内脏器官（图3E）。 </li><li>简单地等待之前退出的needleto减少泄漏的可能性。 </li><li>等待至少两个小时，然后再继续下一步。 </li></ol> 5。鼠标麻醉和鼻内感染<ol><li>准备麻醉在100毫克/公斤和10毫克/公斤，分别使用氯胺酮和甲苯噻嗪。执行的PBS腹腔注射，同型对照抗体，或在所附的书面协议中所述的实验抗体。等待前两个小时后，单克隆抗体注射麻醉鼠标和进行滴鼻感染。 准备使用氯胺酮麻醉在100毫克/公斤和10毫克/公斤，分别为（7）和甲苯噻嗪。<html> </li><li>在下半年的等待期，准备在所附的书面协议中所述的组织胞浆 capsulum接种。 </li><li>按照itens 5.1至5.5。脚趾捏动物镊子和验证反射的情况下，并确认，麻醉工作 。anaesthetization后，轻轻地暂停其门牙鼠标尼龙或棉线。使用2列站，以配合以暂停动物的字符串。 </li><li>慢慢地管理使用一个微管，同时密切监测鼠标的呼吸速率纳惹约50μL接种。让鼠标在这个位置上，以方便酵母沉积到动物的肺（图3F）休息2-5分钟后，鼻内感染。 </li><li>已感染小鼠后，保持在一个监控，温暖的环境（25和37℃之间的温度）鼠标，仔细利用热灯，如果有必要，直到它从麻醉中复苏。当鼠标醒来时，它返回到一个随意获取食物和水的清洁笼笼返回到我们的动物设施，并保持在一个干净的动物房（ BLSI）。 </li></ol> 6。生存研究<ol><li>临床评估的感染和模拟感染的动物，呼吸急促，嗜睡，obtundation和减肥。动物应每天两次检查，实验室成员和动物护理人员每天。 </li><li>小鼠组织胞浆菌病，直到接近生命的尽头，通常是在呼吸率有轻微上升以外的感染没有明显的迹象。凭借先进的疾病，通常发生10-11天，老鼠变得明显tachypneic活动已经减少。通常情况下，他们迅速成为垂死的和过期。 </li><li>仿旧的动物，应适当地与一个专用室中 CO 2安乐死，死去的动物应每天枚举。 </li></ol> 7。 CFU学，组织学和细胞因子的研究<ol><li>每个要收集的器官，准备用10ml PBS 15 ml锥形管。 </li><li>如前所示（5.0 × 10 6）与亚致死inuculum安乐死7和动物感染后14天，并立即删除的肺，脾，肝。 </li><li>取下一块进行评估的机构和执行的固定在福尔马林过夜。该路段将显微镜下的病理评价研究。 </li><li>准备用5ml PBS 50 ml锥形管超过70微米的细胞过滤器，为每个要匀浆器官。浸渍每个机关的剩余部分，​​分别到50毫升管使用无菌5ml注射器活塞。 </li><li>接下来，连续稀释器官匀浆液（1:100，1:1000和1:5000）BHI血琼脂平板（2），每个稀释度的板100μL。 </li><li>根据制造商的指示（罗氏公司，德国）的匀浆加入蛋白酶抑制剂药片。 </li><li>孵育板在37 ° C为14天，并列举CFUs。 </li><li> 4000xg器官组织匀浆离心10分钟，取出器官上清，哗哗将用于在根据制造商的标准方法进行因子分析。 </li></ol> 8。对H.单克隆抗体保护Capsulatum是依赖抗体的特异性和抗体同种型<ol><li>单克隆抗体亚型是对阁下保护的关键capsulatum，以​​HSP60与IgG1的或IgG2a单克隆抗体治疗的小鼠的生存期明显延长，与对照组比较小鼠收到IgG2b单克隆抗体HSP60亚型控制人与生物圈计划或PBS（图4）。 </li><li>此外，收到保护的单克隆抗体在小鼠，在7天的肺和脾的CFU数量显着减少，减少酵母数为2和2.5个对数单位在第14天（图5）肺。 </li></ol><img src="/files/ftp_upload/2532/2532fig1.jpg" alt="图1" /> 图1:H 的增长capsulatum和杂交瘤细胞。 （a）液体火腿的F - 12的文化准备从H.获得单菌落capsulatum一个BHI血琼脂平板上生长。 （二）细胞培养瓶和单克隆抗体纯化蛋白A / G的树脂使用的亲和力cromatography的杂交瘤细胞增长。 <img src="/files/ftp_upload/2532/2532fig2.jpg" alt="图2" /> 图2:H capsulatum滴鼻感染接种的准备。后， 阁下获得的细胞悬液注射器和离心中断capsulatum聚集在血球计数枚举。细胞浓度调整为1.25 × 10 7（生存实验）或5 × 10 6 &lt;50μL（CFUs，细胞因子和组织学） 。 upload/2532/2532fig3.jpg"ALT ="图3"/&gt; 图3:鼠标在单克隆抗体的行政处理。 （一）拿起鼠标的尾巴（B和C），由它的脖子颈背尽量接近，以theears固定。 （四）尾是小指和手掌之间举行。 （五）单克隆抗体的解​​决方案是使用26G1 / 2针注射。 （F）的H. capsulatum接种管理滴鼻，轻轻吹打成一个纳惹细胞悬液。 <img src="/files/ftp_upload/2532/2532fig4.jpg" alt="图4" /> 图4:以HSP60单克隆抗体可以改变组织胞浆菌病的发病机制。 IP注射500微克的IgG1的，或IgG2a单克隆抗体2小时前感染的生存期明显延长（P &lt;0.05，与对照组相比），但一个IgG2b单克隆抗体。 <img src="/files/ftp_upload/2532/2532fig5.jpg" alt="图5" /> 图5:CFU测定肺部在7和14天，后一个亚致死5X10 6 HC小鼠酵母菌治疗用单克隆抗体，以HSP60或不相关的单克隆抗体显示为IgG1的和IgG2a减少真菌的负担，单克隆抗体治疗的动物的IP鼻内挑战。黑条代表在第7天CFUs和白条感染后14天（* P &lt;0.001 7天，** P &lt;0.001），在感染后14天。

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

这里介绍的协议表明，单克隆抗体的到 H capsulatum可以修改实验小鼠组织胞浆菌病的过程中。病原菌的细胞表面抗原的单克隆抗体可以修改主机和病原体之间的相互作用过程中发生的复杂的动态。这项研究建立了单克隆抗体介导腹腔注射时的一个致命性组织胞浆菌病的小鼠模型的保护，并建议候选蛋白疫苗的开发，如H2B，中号抗原（表面过氧化氢酶）（3）和HSP60。管理HC HSP60和H2B保护?...

<table border="1"><tbody><tr><th>材料名称</th><th>公司</th></tr><tr><td>生物安全柜（BSL2） </td><td></td></tr><tr><td> 15 mL锥形管</td><td>猎鹰，BD </td></tr><tr><td>火腿的F - 12培养基</td><td> Gibco公司</td></tr><tr><td> 37 °彗星摇床</td><td></td></tr><tr><td>涡旋</td><td></td></tr><tr><td> 50 ml锥形管</td><td>猎鹰，BD </td></tr><tr><td> 26G1 / 2针</td><td>屋宇署</td></tr><tr><td> 10 mL注射器</td><td>屋宇署</td></tr><tr><td> 250毫升烧瓶</td><td></td></tr><tr><td> FPLC（ 快速蛋白液相色谱法）系统</td><td>通用电气Helthcare </td></tr><tr><td>酶标仪</td><td>百特</td></tr><tr><td>麻醉（氯胺酮和甲苯噻嗪） </td><td></td></tr><tr><td>列代表</td><td></td></tr><tr><td>尼龙绳</td><td></td></tr><tr><td>散热灯</td><td></td></tr><tr><td> 70μm细胞过滤器</td><td>猎鹰，BD </td></tr><tr><td> BHI琼脂平板</td><td> Gibco公司</td></tr></tbody></table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

使用这种方法的目的是1），以推进我们的能力，以保护个人与抗体或疫苗，以防止或治疗由真菌组织胞浆菌和2）审查致病因素的作用，为治疗目标引起的组织胞浆菌病。产生单克隆抗体，免疫小鼠的免疫反应是采用固相酶联免疫吸附在我们的实验室开发的系统进行评估，并选择隔离的脾细胞与杂交瘤细胞融合的最好的响应者小鼠。 C57BL / 6小鼠已被广泛用于研究H。 capsulatum的发病机制，并提供获得所需的数据的最佳模式。为了评估在感染的单克隆抗体的作用，小鼠腹腔要么单抗管理到 H capsulatum或亚型匹配控制单克隆抗体，然后感染要么静脉（IV），腹腔（IP），或滴鼻（中）路线。在科学文献中，单克隆抗体在小鼠体内的真菌感染疗效依赖对死亡率作为终点，在殖民地成蛋白单位（CFU）在较早的时间点评估一起。生存（死亡时间）的研究是必要的，因为他们最能代表人类疾病。因此，我们干预的疗效不会充分地建立无生存曲线。这也是真正建立疫苗疗效或毒力突变体的测试。随着组织胞浆菌病，老鼠经常去充满活力，以死了几个小时。最初可能会干预，如管理一个MAB能力保护的动物疾病，但疾病的复发这不会在短期CFU实验实现。除了生存和真菌的负担分析，我们考察感染（蜂窝招聘，组织学，细胞因子反应）的炎症反应。生存/死亡实验，小鼠被感染，至少每天两次监测发病的迹象。为了评估真菌的负担，组织病理学和细胞因子的反应，小鼠在感染后的不同时期，实施安乐死。根据阿尔伯特爱因斯坦医学院的动物实验研究所的指引，进行动物实验。

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Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL / 6小鼠已被用于研究HC发病机制，并提供最好的模式。我们正在探索这种真菌对体液免疫的潜在利益，并产生了几个单克隆抗体[组蛋白H2B和热休克蛋白60kDa]，我们为他们的腹腔给药后的保护效果测试。

单克隆抗体的反对消极行政 H。 capsulatum和其他真菌病原体

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

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Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

11.9K 次观看.  Albert Einstein College of Medicine. C57BL / 6小鼠已被用于研究HC发病机制，并提供最好的模式。我们正在探索这种真菌对体液免疫的潜在利益，并产生了几个单克隆抗体[组蛋白H2B和热休克蛋白60kDa]，我们为他们的腹腔给药后的保护效果测试。

视频: Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

科研

免疫与感染

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

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 ...