Name: a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)
Uploaded: 2011-09-14T13:00:00
Description: Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

这项工作是由个人从威康信托Beate Kampmann（056608，GRO77273）的奖学金。

1。卡介苗勒克斯记者分枝杆菌股票的制备<ol><li>成长卡介苗LUX（牛分枝杆菌卡介苗蒙特利尔应变穿梭质粒pSMT1的转化）用颤 ​​抖的Middlebrook 7H9肉汤含0.2％甘油，0.05％Tween 80和10％的ADC浓缩在200rpm中旬对数生长期。 </li><li>在光度计试管一式两份（900μlPBS +100μL卡介苗勒克斯文化），准备在无菌PBS 1:10稀释的卡介苗勒克斯文化1毫升。 </li><li>装入管中的N -癸醛（荧光素酶?...

<ol>
	<li>Cheng, S. H., Walker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+increased+anti-mycobacterial+activity+in+peripheral+blood+monocytes+after+BCG+vaccination+in+British+school+children.">Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.</a> Clin Exp Immunol. 74, 20-205 (1988).</li><li>Cheng, S. H., Walker, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+antimycobacterial+activity+before+and+after+Mycobacterium+bovis+BCG+vaccination+in+Chingleput,+India,+and+London,+United+Kingdom.">Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.</a> Infect Immun. 61, 4501-4503 (1993).</li><li>Silver, R. F., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+virulence+of+Mycobacterium+tuberculosis+within+human+monocytes:+virulence+correlates+with+intracellular+growth+and+induction+of+tumor+necrosis+factor+alpha+but+not+with+evasion+of+lymphocyte-dependent+monocyte+effector+functions.">Expression of virulence of Mycobacterium tuberculosis within human monocytes: virulence correlates with intracellular growth and induction of tumor necrosis factor alpha but not with evasion of lymphocyte-dependent monocyte effector functions.</a> Infect Immun. 66, 1190-1199 (1998).</li><li>Hoft, D. F., Worku, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+relationships+between+immune-mediated+inhibition+of+mycobacterial+growth+and+other+potential+surrogate+markers+of+protective+Mycobacterium+tuberculosis+immunity.">Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity.</a> J Infect Dis. 186, 1448-1457 (2002).</li><li>Worku, S., Hoft, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+control+and+antigen-specific+T+cells+on+intracellular+mycobacterial+growth.">Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth.</a> Infect Immun. 71, 1763-1773 (2003).</li><li>Kampmann, B., Gaora, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+human+antimycobacterial+immunity+using+recombinant+reporter+mycobacteria.">Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.</a> J Infect Dis. 182, 895-901 (2000).</li><li>Cheon, S. H., Kampmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+activity+in+whole+blood+as+a+potential+surrogate+marker+of+immunity+after+vaccination+against+tuberculosis.">Bactericidal activity in whole blood as a potential surrogate marker of immunity after vaccination against tuberculosis.</a> Clin Diagn Lab Immunol. 9, 901-907 (2002).</li><li>Janulionis, E., Sofer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+activity+of+orally+administered+clofazimine+against+intracellular+Mycobacterium+tuberculosis+in+whole-blood+culture.">Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture.</a> Antimicrob Agents Chemother. 48, 3133-3135 (2004).</li><li>Kampmann, B., Tena, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+human+in+vitro+system+for+evaluating+antimycobacterial+vaccines.">Novel human in vitro system for evaluating antimycobacterial vaccines.</a> Infect Immun. 72, 6401-6407 (2004).</li><li>Kampmann, B., Tena-Coki, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+antimycobacterial+immune+responses+in+HIV-infected+children+receiving+HAART.">Reconstitution of antimycobacterial immune responses in HIV-infected children receiving HAART.</a> Aids. 20, 1011-1018 .</li><li>Martineau, A. R., Wilkinson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dose+of+vitamin+D+enhances+immunity+to+mycobacteria.">A single dose of vitamin D enhances immunity to mycobacteria.</a> Am J Respir Crit Care Med. 176, 208-213 (2007).</li></ol>

在结核病疫苗研究中的杀菌检测以及建立动物模型，它们应用到屏幕上可能有前途的候选疫苗。作为主要的免疫原性读出各个器官功能的细菌负荷减少。 人类杀菌实验的第一代设计单独使用巨噬细胞，感染与 M. 结核病 。 CFU担任分枝杆菌生存的主要读出后最短的时间为3周。依靠这些检测外周血单个核细胞，贴壁的单核细胞/巨噬细胞和裂解准备在特定时间点<...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫没有 </td></tr><tr><td>卡介苗勒克斯 </td><td>各种</td><td> N / A </td></tr><tr><td> Middlebrook 7H11琼脂</td><td>碧迪</td><td> 283810 </td></tr><tr><td> Middlebrook 7H9肉汤</td><td> BD公司</td><td> 271310（500克） </td></tr><tr><td> Middlebrook ADC的浓缩</td><td> BD公司</td><td> 212352 </td></tr><tr><td> Middlebrook OADC的补充</td><td>碧迪</td><td> 212240 </td></tr><tr><td>吐温80 </td><td>西格玛爱秩序</td><td> 274364 </td></tr><tr><td>甘油</td><td>西格玛爱秩序</td><td> G5150 </td></tr><tr><td>潮霉素B </td><td>罗氏公司</td><td> 10843555001（20毫升） </td></tr><tr><td> N -癸醛</td><td>西格玛</td><td> D7384 - 100G </td></tr><tr><td>乙醇（&gt; 99.7％） </td><td> VWR </td><td> 101077 </td></tr><tr><td>无菌PBS </td><td>众议院</td><td> N / A </td></tr><tr><td> RPMI - 1640 L -谷氨酰胺，25MM的HEPES </td><td>西格玛</td><td> R0883 </td></tr><tr><td>无菌蒸馏水</td><td>众议院</td><td> N / A </td></tr><tr><td colspan="3"> 设备 </td></tr><tr><td>管光度计（强制注射端口） </td><td>贝特霍尔德AutoLumat LB953或单管光度计天狼星</td><td></td></tr><tr><td>光度计管，12 × 75毫米，耐热无框</td><td>康宁从VWR </td><td> 99445-12 </td></tr><tr><td>通风帽的锥形瓶，无菌聚碳酸酯250毫升</td><td>康宁通过费舍尔科学</td><td> 2150329 </td></tr><tr><td> BD真空采血管血液收集管（不含防腐剂肝素） </td><td>屋宇署</td><td> 367676 </td></tr><tr><td> 50ml隼管中，圆锥形</td><td>屋宇署</td><td> 352077 </td></tr><tr><td> 7毫升Sterilin标本聚丙烯茹管</td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin环球货柜（30毫升） </td><td>实验室分析有限公司</td><td> 128C </td></tr><tr><td>玻璃珠直径2mm </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10毫升移液器和移液器男孩</td><td>任何</td><td></td></tr><tr><td> 1000μl和200μL的移液器和过滤嘴</td><td>任何</td><td></td></tr><tr><td colspan="3"> 通用实验室设备： </td></tr><tr><td> II级安全柜柜</td><td></td><td></td></tr><tr><td>冰箱（4℃） </td><td></td><td></td></tr><tr><td>离心分离</td><td></td><td></td></tr><tr><td>混合管的摇摆振动筛平台（180 0 ） </td><td></td><td></td></tr><tr><td>长大细菌培养轨道摇床培养箱在37 ° C </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

功能测定早已起到了关键作用的一个给定的疫苗的免疫原性的测量。这是常规表现为血清杀菌滴度。儿童疫苗的血清杀菌滴度的研究使我们能够开发和验证切断保护性免疫反应的水平和削减权衡，在日常使用。没有这样的检测已采取迈进的诱导主要是细胞免疫效应T细胞反应的形式，如结核病疫苗，疫苗的例行评估。表现一个给定的候选疫苗在动物模型，定期评估标准化杀菌检测，目前所有的新型结核病疫苗候选人受到这一步在他们的评价，第1阶段的人体试验之前。理想的免疫原性，因此，新型抗结核病疫苗的保护效力的可能性的评估应在人体模型进行类似的分步进行的评价，现在，包括杀菌实验的测量。 杀菌检测在结核病疫苗研究方面已经建立的动物模型，它们应用到屏幕上可能有前途的候选疫苗。作为主要的免疫原性读出各个器官功能的细菌负荷减少。然而，没有这样的检测已被纳入临床试验新型抗结核病疫苗的最新。 虽然仍存在不确定性的确切机制，导致人类巨噬细胞内结核分枝杆菌杀，分枝杆菌与巨噬细胞和T细胞的相互作用是明确要求。本文中所描述的实验代表的新型杀菌检测等关键的细胞成分的研究，使全血中目前没有作出有关其相对独立的贡献的假设与所有其他细胞和体液因素。本集团所描述的检测使用全血的小卷，并已受聘在成人和儿童的研究结核病流行的设置。我们已经表明BCG疫苗的免疫原性，增加分枝杆菌的增长，在HIV阳性患者，以及抗逆转录病毒疗法和维生素 D分枝杆菌在体外生存的影响。在这里，我们总结的方法，目前我们的重复性数据，使用这个相对简单，低成本和友好的领域模型。 注：定义/缩写卡介苗勒克斯= M 牛卡介苗，蒙特利尔应变，转化与穿梭质粒进行哈维氏弧菌的luxAB基因pSMT1，分枝杆菌GroEL的（HSP60）启动子的控制下。 CFU =集落形成单位（结核分枝杆菌的可行性措施）。

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

我们描述了一个替代的方法来列举分枝杆菌<em&gt;在体外</em&gt;，它使用的记者基因标记，而不是细胞集落形成单位（CFU）的分枝杆菌。 “生存”的生物体，以及宿主反应的标记，同时测量，提供一个低成本，灵活和功能系统在结核病中的主机/病原体相互作用的研究。

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Research

JoVE Journal

Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Use of a Monocyte Monolayer Assay to Evaluate FcÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Receptor-mediated Phagocytosis

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...