Name: quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays
Uploaded: 2011-04-04T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays at JoVE.com

作者想感谢夫人莫尼卡Tocchi编辑对稿件的审查。支持AMH是由阿卜杜勒阿齐兹国王大学的奖学金，在加拿大通过沙特阿拉伯文化局。

1。制备试剂和仪器<ol><li>在开始狭缝杂交过程之前，准备的Tris缓冲液（20毫米的Tris，137 mM氯化钠，pH值7.6）公司（TBS）的血凝素，或房委会，狭缝杂交，或0.01％20_mls Zwittergent 4M尿素溶液20_mls在TBS的神经氨酸酶，或NA，狭缝杂交的解决方案。 4M尿素要新鲜，每次准备，10％的DH 2 O Zwittergent原液稳定，在室温下至少6个月，因此可以被摊薄的TBS每次检测前的0.01％。 </li><li>准备2000毫升的洗涤?...

<ol>
	<li>Webster, R. G., Bean, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+influenza+virus.">Genetics of influenza virus.</a> Annu Rev Genet. 12, 415-431 (1978).</li><li>Skehel, J. J., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+membrane+fusion+in+virus+entry:+the+influenza+hemagglutinin.">Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.</a> Annu Rev Biochem. 69, 531-569 (2000).</li><li>Wood, J. 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流感病毒的HA和NA的定量测定疫苗的研究和开发的关键，因为这两个表面蛋白是最重要的诱导免疫反应6-11的病毒成分。此前报道的免疫学方法对这些蛋白质的检测需要应变的特异性抗体。简单，重复性好，快速的狭缝杂交的方法来量化这里描述的HA和NA抗原是适用于所有A型流感病毒的HA和NA蛋白抗体识别只有普遍保守的抗原决定簇 6-8 。在这个方法感兴趣的调查的唯一的困难可能是?...

An author's affiliation was omitted from the publication of <a href="http://www.jove.com/video/2784">Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays</a>.

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

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>生物点SF微滤器械</td><td> Bio - Rad公司</td><td> 170-6542 </td><td></td></tr><tr><td>生物点SF过滤纸</td><td> Bio - Rad公司</td><td> 162-0161 </td><td></td></tr><tr><td>的Immobilon - FL转印膜（聚偏氟乙烯） </td><td> Millipore公司</td><td> IPFL00010 </td><td></td></tr><tr><td>真空泵</td><td> Millipore公司</td><td> WP6111560 </td><td></td></tr><tr><td>化学发光百玛士光膜</td><td>柯达</td><td> 178 8207 </td><td></td></tr><tr><td>氟化凝胶文件系统</td><td>阿尔法INNOTECH公司</td><td> 29 - 008 - 1896X </td><td></td></tr><tr><td>针对HA和NA抗原的通用兔抗体</td><td></td><td> UNI - 1（“房委会”）HCA - 2，HCA - 3（NA） </td><td>抗体可通过MTA或有兴趣的研究者可以根据先前所描述的6,7的程序产生。 </td></tr><tr><td>流感疫苗参考抗原</td><td> CBER / FDA或NIBSC </td><td></td><td></td></tr><tr><td>流感疫苗样本</td><td></td><td></td><td>在大多数国家普遍使用</td></tr><tr><td>尿素</td><td> Sigma - Aldrich公司</td><td> U1250 </td><td></td></tr><tr><td> Zwittergent 3-14洗涤剂</td><td> Calbiochem </td><td> 693017 </td><td></td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337 - 500 </td><td></td></tr><tr><td>杂交级拦截器非脱脂奶粉</td><td> Bio - Rad公司</td><td> 170-6404 </td><td></td></tr><tr><td> ImmunoPure山羊抗兔IgG（H + L），过氧化物酶</td><td> Thermo Scientific的</td><td> 31460 </td><td></td></tr><tr><td> SuperSignal西DURA工期顺延基板</td><td> Thermo Scientific的</td><td> 34075 </td><td></td></tr></tbody></table>

quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays

血凝素（HA）和神经氨酸酶（NA）是流感病毒，这是众所周知的发挥重要作用，在病毒的生命周期和诱导免疫反应的保护1,2两个表面蛋白。由于中和抗体的主要目标，房委会是目前使用的流感疫苗的效力标记和测量单向免疫扩散法（SRID）3 。然而，提供相应的亚型特异性血清的SRID的依赖，导致延迟2-3个月的最低，为每一个新的疫苗版本。此外，尽管有证据表明，娜也诱导保护性免疫，NA的流感疫苗数量，由于缺乏适当的试剂或分析方法5尚不规范。因此，简单的量化HA和NA抗原的替代方法是可取的快速释放和更好的流感疫苗的质量控制。 在所有可用的A型流感HA和NA序列普遍保守区域确定了生物信息学分析6-7。只有在普遍保守的HA，抗原表位的融合肽的6的，而两个保守序列neuraminidases，一靠近酶的活性部位（HCA - 2）和一个序列（UNI - 1）被确定其他接近的N -端（HCA - 3）7。这些氨基酸序列的肽合成和用于免疫家兔抗体的产生。对房委会的UNI - 1的抗原表位的抗体能够绑定到13亚型的A型流感HA（H1 - H13），而对HCA - 2和HCA - 3地区的NA的抗体能够有约束力的全部9个NA亚型。所有的抗体对检测尿囊蛋白无交叉反应的观察证明，病毒序列显示了显着的特异性。这些普遍的抗体，然后用于发展狭缝杂交分析，量化而不需要特定抗血清 7,8的HA和NA在A型流感疫苗。疫苗样本应用到PVDF膜的使用以及与不同浓度稀释到的参考标准插槽印迹装置。医管局检测，样品和标准，先稀释在Tris缓冲液（TBS）含有4M尿素，而NA的测量他们的TBS稀释含有0.01％Zwittergent这些条件显着提高了检测的灵敏度。按照各自的普遍抗体免疫印迹的HA和NA抗原的检测，密度测量信号强度进行了量化。 HA和NA在疫苗的数额，然后使用标准曲线的建立与使用引用不同浓度的信号强度计算。 鉴于这些抗体结合在HA或NA普遍抗原表位，有兴趣的研究者可以作为研究工具使用，他们在其他免疫仅比狭缝杂交。

Watch this Scientific Journal Video about Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays at JoVE.com

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

一个简单的狭缝杂交方法被开发用于流感病毒的血凝素和使用针对其最保守的序列，通过生物信息学分析确定的普遍抗体神经氨酸酶的量化。这种创新的方法可以提供一个有用的替代所有病毒的血凝素和神经氨酸酶的定量测定。

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 ...

Research

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