Name: measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells
Uploaded: 2017-11-22T15:00:00
Description: Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

我们感谢 Dr. 绣花卢, Dr., 和 Ms. 从疾病预防控制中心的流感部门的阿什利。我们感谢 CDC 流感部门的 Dr. 利伯, 帮助他准备图 3的图形。最后, 我们感谢 Dr. m Matrosovich, 马尔堡, 德国提供 MDCK-SIAT1 细胞。

所有流感病毒都应按照微生物和生物医学实验室生物安全 (BMBL)12所界定的适当的生物安全水平要求 (BSL-2 或更高) 来处理。

1. 试剂和起动材料的制备
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制备 MDCK-SIAT1 细胞和无菌细胞培养基
	<ol>
<li>使用500毫升 Dulbecco 的改良鹰培养基 (DMEM) 制备具有高葡萄糖、10% v/v 热灭活胎牛血清 (FBS)、2毫米 l-谷氨酰胺、1 mM 丙酮酸钠、1毫克/毫升 G418 ...

<ol>
	<li>Reber, A., Katz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+assessment+of+influenza+vaccines+and+immune+correlates+of+protection.">Immunological assessment of influenza vaccines and immune correlates of protection.</a> Expert Rev of Vaccines. 12, 519-536 (2013).</li><li>Li, C. K., Rappuoli, R., Xu, X. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+immunogenicity+of+influenza+viruses+cultivated+in+Vero+or+MDCK+cells+and+in+embryonated+chicken+eggs.">Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.</a> Dev Biological Stand. 98, 39-51 (1999).</li><li>Levine, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+Antibody+Responses+to+Antigenically+Drifted+Influenza+A(H3N2)+Viruses+among+Children+and+Adolescents+following+2014-2015+Inactivated+and+Live+Attenuated+Influenza+Vaccination.">Neutralizing Antibody Responses to Antigenically Drifted Influenza A(H3N2) Viruses among Children and Adolescents following 2014-2015 Inactivated and Live Attenuated Influenza Vaccination.</a> Clin Vaccine Immunol : CVI. 23, 831-839 (2016).</li><li>Lin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characteristics+and+antigenic+properties+of+recently+emerged+subclade+3C.3a+and+3C.2a+human+influenza+A(H3N2)+viruses+passaged+in+MDCK+cells.">The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells.</a> Influenza Other Respir Viruses. , (2017).</li><li>Lin, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuraminidase+receptor+binding+variants+of+human+influenza+A(H3N2)+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site:+a+role+in+virus+attachment?.">Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?.</a> J of Virol. 84, 6769-6781 (2010).</li><li>Skowronski, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+acquired+during+cell+culture+isolation+may+affect+antigenic+characterisation+of+influenza+A(H3N2)+clade+3C.2a+viruses.">Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses.</a> Euro Surveillance. 21, 30112 (2016).</li><li>Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N. A., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+alpha-2,6-sialyltransferase+in+MDCK+cells+increases+influenza+virus+sensitivity+to+neuraminidase+inhibitors.">Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.</a> J of Virol. 77, 8418-8425 (2003).</li><li>Oh, D. Y., Barr, I. G., Mosse, J. A., Laurie, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDCK-SIAT1+cells+show+improved+isolation+rates+for+recent+human+influenza+viruses+compared+to+conventional+MDCK+cells.">MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells.</a> J Clin Microbiol. 46, 2189-2194 (2008).</li><li>US Department of Health and Human Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2009).</li><li>Reed, H. 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锰测定法是流感血清学中用于检测流感感染或疫苗接种后抗体应答的主要方法之一。由锰测定产生的滴常被用作许多流感血清研究的主要结果。锰测定法也广泛用于血清诊断和疫苗免疫原性评价。国际 inter-lab 的研究已经进行比较, 在多个实验室的锰化验结果14。
与 HI 相比, 锰测定法的目的是直接测量细胞培养中能中和病毒感染的功能性抗体。世界各地的野?...

流感病毒每年继续造成人类人口的发病率和死亡率。HA 是流感病毒的主要表面糖蛋白。中和抗体靶向 HA 是主要的免疫机制, 与流感感染的保护相关的1,2。血凝抑制 (HI) 和锰测定法是广泛用于检测流感病毒感染或疫苗接种后的人血清中抗体应答的两种方法3。高含量的检测方法可抑制红细胞的病毒血凝, 并被认为是一种替代性试验。与 HI 不同, 锰测定法可以直接测定人血清中的抗体水平, 从而消除细胞培养中的流感感染。MDCK 细胞常用于流感病毒分离和锰含量测定4。
流感病毒经常进行抗原漂移和转移, 获取 HA 蛋白的突变, 从而改变病毒的受体结合特异性。自2014年以来, 新的一组 (H3N2) 病毒出现并继续流传到本赛季。这些病毒大多数属于基因组 3 c. 2 a 和 3 c. 3 a 基于 HA 蛋白质的系统进化分析。许多循环 3 c. 2 的病毒已经降低了 hemagglutinate 红细胞的能力, 因此不能用 hi 化验5来表征。中和测定必须用于测量这些病毒的抗体应答, 而不 hemagglutinate6。此外, 研究表明, 这些当代 a (H3N2) 病毒改变受体结合性能与早期 a (H3N2) 病毒, 并倾向于积累文化适应突变和多态性时, 传代在体外细胞区域性7,8,9。与传统的 MDCK 细胞相比, MDCK-SIAT1 是由 Matrosovich et al.开发的细胞系。通过α2,6-sialtransferase (SIAT1) 的 cDNA MDCK 细胞的稳定转染。这条细胞线表达了增加的α2,6-唾液半乳糖基和减少α2,3-唾液酸基的数量比父 MDCK 细胞10。与 MDCK 细胞11相比, MDCK-SIAT1 细胞已显示出改善 (H3N2) 病毒的隔离率。最近, 林et al.报告说, 对于新出现的 3 c. 2 a 和 3 c. 3 a 人 a (H3N2) 流感病毒, 更忠实的病毒复制和更好的病毒传染, 当病毒被培养了在 MDCK-SIAT1 细胞线与 MDCK 细胞对比7。因此, MDCK-SIAT1 细胞更适合于锰测定, 以表征抗体对最近一组 (H3N2) 病毒的反应。
在这里, 我们描述了一个使用 MDCK-SIAT1 细胞的锰测定方法来测量当代 3 c. 2 a 和 3 c. 3 a a (H3N2) 病毒在人类血清中的抗体应答。在这种方法中, 可以使用在卵或细胞中生长的病毒。含有中和抗体的热灭活血清先被连续稀释, 然后用 100 TCID50/甲型流感病毒 (H3N2) 进行孵育, 使血清中的抗体与病毒结合。然后将 MDCK-SIAT1 细胞添加到病毒抗体混合物中, 在37° c 下孵育 18-20 h, 5% CO2 , 允许 A (H3N2) 病毒感染 MDCK-SIAT1 细胞并进行复制。经过一夜的孵化, 该板块是固定和数量的病毒在每个井是量化的 ELISA 使用流感的一个 NP 单克隆抗体。NP 的检测表明病毒感染的存在和中和抗体的缺乏。中和抗体滴度被定义为最高的血清稀释的倒数, 提供了≥50% 抑制病毒传染性。

<table border="0" cellpadding="0" cellspacing="0" width="969">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM) with high Glucose</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">11965</td>
			<td style="width:308px;">A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal bovine serum (FBS)</td>
			<td style="width:181px;">Hyclone</td>
			<td style="width:185px;">SH30070.03</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Bovine Serum Albumin (BSA) Fraction V, Protase Free</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">3117332001</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">L-Glutamine</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">25030-081</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium pyruvate</td>
			<td style="width:181px;">Life Science</td>
			<td>11360-070</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Geneticin G-418 disulfate salt&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">A1720-5G&#160;&#160;</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">HEPES</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15630-080</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/Streptomycin</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15140-122</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:295px;">Acetone</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">67-64-1</td>
			<td style="width:308px;">Used at an 80% concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Phosphate-Citrate Buffer with Sodium Perborate&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBF2806V</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">O-Phenylenediamine Dihydrochloride tablet</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBQ1086V</td>
			<td style="width:308px;">1 tablet per 100ml of cell culture grade water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:295px;">Sulfuric Acid</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:185px;">A510-P500</td>
			<td style="width:308px;">Used 0.5M final concentration</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">Ethanol, Denatured, 4L</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">EM-AX0422-3</td>
			<td style="width:308px;">Used at an 70% concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Trypsin-EDTA</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">1748048</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RDE II &#34;Seiken&#34;</td>
			<td style="width:181px;">Denka Seiken</td>
			<td style="width:185px;">370013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tween 20</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">P1379-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-NP mouse monoclonal Ab</td>
			<td style="width:181px;">Millipore pool</td>
			<td style="width:185px;">MAB 8257 MAB 8258</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-mouse IgG HRP&#160;</td>
			<td style="width:181px;">KPL</td>
			<td style="width:185px;">074-1802</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">96-well flat-bottom plates</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:185px;">3455</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Plate reader</td>
			<td style="width:181px;">Molecular Device</td>
			<td style="width:185px;">Spectromax 384 plus</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell Culture Flask 162 cm2/Vent Cap</td>
			<td style="width:181px;">Corning/VWR</td>
			<td style="width:185px;">3151</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells

对流感病毒血 (HA) 的中和抗体被认为是与保护流感感染相关的主要免疫机制。Microneutralization (MN) 检测通常用于测量流感疫苗接种或感染后人血清中和抗体的反应。机达比犬肾脏 (MDCK) 细胞是常用的细胞基质的锰测定方法。然而, 目前循环 3 c. 2 a 和 3 c. 3 a a (H3N2) 流感病毒已获得改变受体结合特异性。MDCK-SIAT1 细胞系增加α-26 唾液半乳糖基在表面上已证明提供改善感染和更忠实的复制比传统的 MDCK 细胞为这些当代 A (H3N2) 病毒。在这里, 我们描述了使用 MDCK-SIAT1 细胞的锰测定方法, 它已经被优化, 以量化中和抗体滴到这些当代 a (H3N2) 病毒。在本协议中, 含有中和抗体的热灭活血清首先依次稀释, 然后用 100 TCID50/流感病毒 A (H3N2) 细菌孵育, 使血清中的抗体与病毒结合。然后将 MDCK-SIAT1 细胞添加到病毒抗体混合物中, 在37° c 下孵育 18-20 h, 5% CO2 , 允许 (H3N2) 病毒感染 MDCK-SIAT1 细胞。在夜间孵化后, 用流感 A 核 (NP) 单克隆抗体的酶联免疫吸附试验 (ELISA) 对每一个井中的病毒数量进行固定和定量。中和抗体滴度被定义为最高的血清稀释的相互作用, 提供50% 抑制病毒传染性。

测定病毒种群的传染性是锰测定的第一步。图 2说明了用于确定病毒储存的 TCID50的版面布局。对于未知传染性的病毒种群, 病毒可以从多个 pre-dilutions 滴定, 例如 10-2和 10-3, 以便捕获最佳的滴定曲线来计算病毒的传染性。在锰测定中使用的病毒数量应标准化到 100 TCID50/井 (50 µL)。病毒储存的最小稀释量达到 100 TCID50...

Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

流感中和抗体与流感感染的保护相关。Microneutralization 测定检测人血清中和抗体, 通常用于流感人血清学。我们描述了一个 microneutralization 的方法, 使用 MDCK-SIAT1 细胞测量中和抗体滴的当代 3 c. 2 a 和 3 c. 3 a a (H3N2) 病毒在流感疫苗接种或感染后。

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

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Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

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免疫与感染

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

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 ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal 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 ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...