Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

该项目的经费部分来自国家过敏和传染性疾病研究所、国家卫生研究所、卫生和人类服务部的联邦基金, CEIRS 合同 P01AI097092-04S1 (P.E.L.)。

本手稿中所做的实验是按照伊坎医学院的《道德和法规规范》进行的。
1. 通过转染或感染来表达流感病毒血凝素
染:
<ol><li>平板人胚胎肾脏 (HEK 293T) 细胞密度为 2 x 104细胞/在白色组织培养中处理96井板, 让它在4个°C 孵化器 (与 37 CO5%) 坐在 2 h。</li>
	<li>染细胞与 100 ng DNA 编码的病毒血凝素和 0.2 ul 的转染试剂每井?...

<ol>
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此处描述的方法允许用户测量 HA 特定单克隆抗体与小鼠 FcγRIV 的接触能力。靶抗原、流感病毒血凝素, 在病毒感染或质粒 DNA 转染后在细胞表面表达。这项试验还可以与其他 FcγRs (人类或小鼠) 结合使用不同的表面表达的病毒蛋白。此外, 我们使用血清样本来评估抗体的能力, 在多克隆的情况下, 以诱发 Fc 效应函数 (数据未显示), 这可能提供更多的相关信息, 当研究感染诱发的适应性免疫反应或接种....

季节性流感疫苗提供的免疫传统上是通过血凝抑制 (HI) 检测方法1来评估的, 它测量了针对血凝素受体结合部位的抗体的存在。这些高活性抗体可以赋予杀菌免疫, 但在其保护范围内一般是狭窄的, 只对少数几株流感病毒提供免疫力。广泛反应性单克隆抗体的分离和鉴定血凝素 (HA) 表明, 普遍流感疫苗的发展范围内可达2, 3, 4,5,6,7,8. 普遍流感疫苗的主要目标之一是对流感病毒的保守区域(9、10、11、12 )产生强烈的抗体应答。,13,14,15,16. 有广泛的反应性抗体识别这些保守的表位, 由中和和非中和抗体17,18, 被证明需要 Fc FcγR 的相互作用, 以优化保护在体内并突出显示 Fc 介导的免疫对流感病毒的整体免疫应答的贡献19,20。
保护的相关性对于评估感染的免疫力至关重要。这些指标允许科学家和临床医生评估疫苗的功效、数据的重要性和治疗过程。预防流感病毒感染的唯一确定的相关性是血凝抑制试验。1:40 的滴度与患病风险的50% 降低有关 (121 ), 并反映了抗体的存在, 通过靶向位于 HA 球状头的受体结合点来抑制凝集。然而, 也应该指出, T 细胞反应可能是更好的相关性预防流感病毒感染的老年人。有趣的是, 最近的一份报告表明, 神经氨酸酶抑制活性可能是更好的预测流感免疫的22。幸运的是, 典型的体外检测方法, 如中和或神经氨酸酶抑制试验可以测量 HA 茎或神经氨酸酶特异抗体。然而, 大多数这些常规的体外化验只考虑抗体的抗原结合区的功能, 而不测量 Fc 区域的作用。此外, 未检测到通过 Fc 受体参与在体内中保护的非中和抗体的贡献17,18。为了测量诱发 Fc 介导免疫的抗体所提供的保护, 如抗体依赖性细胞介导的毒性 (ADCC), 需要一个健壮的体外检测。
下面描述的方法评估小鼠单克隆抗体的能力, 通过使用基因修饰的 Jurkat 细胞系, 表达激活鼠型 1 FcR, FcγRIV, 诱导 Fc 介导的功能。抗体参与 FcγR transduces 细胞内信号, 触发核因子的活化 T 细胞介导的荧光素酶活性。该方法优于传统技术, 需要分离和培养原效应细胞, 使用流式细胞仪检测 Fc 介导的效应函数的激活19,23,24 ,25,26。首先, 这里描述的协议可以很容易地被改编成包含不同病毒的目标, FcγRs 和不同物种 (人和小鼠) 的抗体。其次, 使用修饰的 Jurkat 细胞系表达 FcγR 与荧光素酶报告基因在活化 T 细胞 (NFAT) 核因子下, 允许一个大的格式分析, 可以很容易地用一个平板阅读器测量发光。在这里, 传统的激活的主要效应细胞被取代诱导 NFAT-荧光素酶的抗体接触 FcγR 表面上的 Jurkat 细胞。这96井板格式化验允许测量多达四个不同的样品在 triplicates 与七稀释-许多样品可能是繁琐的使用传统技术。另外还有一点需要考虑, 这可能会影响实验设计和数据的解释。病毒靶必须在细胞表面表达, 以便抗体识别。为了减轻这种情况, 目标抗原 (例如内部病毒蛋白) 可以直接涂在盘子的表面上。然而, 这还没有经过严格的测试。此外, 修改后的 Jurkat 细胞系只表达一种激活 FcγR 的类型, 不表达任何抑制 FcγRs, 而主细胞系表达所有受体在生理环境中。
我们以前用这种方法来证明多克隆语境中的表位特异性在调节 Fc 介导的效应函数方面起着关键作用, 而抗体依赖性细胞介导反应的最佳活化需要两点联系27,28。在这里, 我们描述一个方法, 评估的能力, 茎特定单克隆抗体参与和激活小鼠激活 FcγRIV。尽管有异常29、30, 但大量广泛的反应性抗体瞄准了血凝素的抗原保守的茎区, 从而在普遍流感的发展中起着重要作用。疫苗.

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			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

抗体通过其抗原结合域和 Fc 区域, 在耦合病毒病原体的先天和适应性免疫应答中起着至关重要的作用。在这里, 我们描述如何测量 Fc 效应函数的活化作用的单克隆抗体靶向流感病毒血凝素与使用基因工程的 Jurkat 细胞系表达活化型 1 Fc FcγR. 使用此方法,免疫球蛋白所赋予的特定 Fc-FcγR 相互作用的贡献可以通过体外测定来确定。

我们证明, 一个茎特定的单克隆, 6F12, 但不是头部特异的单克隆, PY102, 能够激活主要的自然杀伤细胞植入 CD107a 和干扰素γ19。为了模拟茎特异性抗体激活原发性 NK 细胞的能力, 我们表明, 一个茎特异性的单克隆抗体, 6F12, 可以有力地激活修饰的 Jurkat 细胞, 表达小鼠 FcγRIV 14 倍。另一方面, 头部特定的单克隆抗体, PY102 和对照 IgG 不 (图 2)28。</p...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

我们描述了一种方法来衡量 Fc 介导的效应函数的活化作用的抗体, 针对流感病毒血凝素。这种检测也可以适应评估单克隆抗体或多克隆血清靶向其他病毒表面糖蛋白, 以诱导 Fc 介导的免疫能力。

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Research

JoVE Journal

Immunology and Infection

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

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

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

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 ...