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。

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作者报告没有利益冲突。这份出版物的调查结果和结论是作者的, 不一定代表疾病控制和预防中心和资助机构的观点。

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。世界各地的野外实验室都有各种形式的锰测定方法。出的分析 (即, 减少在中和抗体存在下的病毒感染) 可以基于 ELISA 定量的病毒 NP 抗体, HA 定量的病毒, 或免疫染色的病毒斑块在每个井在细胞培养中存在抗体和潜伏期后感染病毒14,15。各种形式的 fluorescent-based 斑块还原中和试验 (例如, 聚焦还原法和 ViroSpot 测定) 通常用于大量流感病毒场分离株的抗原鉴定, 部分原因是低病毒这些化验所需的金额15,16。为了描述人类血清学中流感抗体的反应, 世界卫生组织 (世卫组织) 全球流感监测网络建议采用2天的 ELISA 法进行血清诊断, 以检测流感3。该方法也广泛应用于血清流行病学研究和流感疫苗接种后抗体应答的评估。它主要检测到流感 HA 表面蛋白的抗体, 因此可以检测功能性菌株特异抗体。
在这里, 我们描述了一个2天的 ELISA-based 锰测定, 利用 MDCK-SIAT1 细胞。这一方法的优化, 以检测中和抗体的当代 A (H3N2) 病毒的人血清。使用 MDCK-SIAT1 细胞进行锰含量测定时应考虑几个关键步骤。首先, MDCK-SIAT1 细胞应在含有 G418 硫酸盐的培养基中传代, 以维持人αSIAT1 cDNA 在细胞系中的稳定性。在使用 MDCK-SIAT1 细胞进行病毒传播和锰含量测定时, 介质中不需要 G418 硫酸盐 (例如, g-418)。在锰含量测定中使用的细胞应在记录生长阶段, 75-95% 70-100。其次, 良好的病毒储存是必要的, 以便进行成功的锰化验。病毒类股应具有高传染性, 由 TCID50滴定和最小数量的缺陷颗粒测量。病毒储存达到 100 TCID50/井病毒的最低稀释量应等于或大于1:100。虽然卵和细胞传代病毒都可以用于锰的检测, MDCK-SIAT1 细胞线保持更好的遗传稳定性, 传播 3 c. 2 a 和 3 c. 3 a a (H3N2) 病毒7。在病毒传播后, 应对病毒种群的 HA 和 NA 基因进行测序, 以确保在关键抗原部位不引入任何卵细胞或细胞培养适应突变, 这可能会导致检测中使用的病毒的抗原性发生变化。第三, 每种化验中所使用的传染性病毒的数量应仔细滴定, 并在每种化验结果中加入一种反滴定法来验证。检测中使用的病毒过多可能会降低抗体的滴, 降低测定的灵敏度。同样, 检测中使用的病毒过少也会导致 VC 信号弱、背景 (CC) 和假阳性结果。因此, 重要的是要包括适当的阳性和阴性控制血清的检测, 以监测性能。当比较使用同一血清的多种病毒的抗体应答时, 确保所有病毒的背部滴定在一个可接受的范围内是至关重要的。
用 MDCK-SIAT1 细胞进行锰含量测定是灵敏的, 特异的检测抗体对当代 3 c. 2 a 和 3 c. 3 a a (H3N2) 流感病毒在人类血清中, 包括抗原漂移 a (H3N2) 病毒6。该方法已被用于评估接种的人血清面板 3 c. 2 a 和 3 c. 3 a a a (H3N2) 病毒在灭活流感疫苗接种以后5,17,18。然而, 如流感病毒继续获得突变, 可以改变抗原性和受体结合性质的病毒和引起抗原漂移, 需要不断努力, 以优化现有的流感血清学化验, 以保持新出现的流感病毒所需的敏感性和特异性。

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><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM) with high Glucose</td><td>Life Science</td><td>11965</td><td>A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) Fraction V, Protase Free</td><td>Sigma-Aldrich</td><td>3117332001</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Science</td><td>25030-081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Science</td><td>11360-070</td><td></td></tr><tr><td>Geneticin G-418 disulfate salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A1720-5G&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>HEPES</td><td>Life Science</td><td>15630-080</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Science</td><td>15140-122</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>67-64-1</td><td>Used at an 80% concentration</td></tr><tr><td>Phosphate-Citrate Buffer with Sodium Perborate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SLBF2806V</td><td></td></tr><tr><td>O-Phenylenediamine Dihydrochloride tablet</td><td>Sigma-Aldrich</td><td>SLBQ1086V</td><td>1 tablet per 100ml of cell culture grade water</td></tr><tr><td>Sulfuric Acid</td><td>Fisher Scientific</td><td>A510-P500</td><td>Used 0.5M final concentration</td></tr><tr><td>Ethanol, Denatured, 4L</td><td>VWR</td><td>EM-AX0422-3</td><td>Used at an 70% concentration</td></tr><tr><td>Trypsin-EDTA</td><td>Life Science</td><td>1748048</td><td></td></tr><tr><td>RDE II &quot;Seiken&quot;</td><td>Denka Seiken</td><td>370013</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379-500ml</td><td></td></tr><tr><td>Anti-NP mouse monoclonal Ab</td><td>Millipore pool</td><td>MAB 8257 MAB 8258</td><td></td></tr><tr><td>Anti-mouse IgG HRP&amp;nbsp;</td><td>KPL</td><td>074-1802</td><td></td></tr><tr><td>96-well flat-bottom plates</td><td>Thermo Scientific</td><td>3455</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Device</td><td>Spectromax 384 plus</td><td></td></tr><tr><td>Cell Culture Flask 162 cm2/Vent Cap</td><td>Corning/VWR</td><td>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/井是1:100。
 图 3描述使用 MDCK-SIAT1 单元进行锰测定的关键步骤。热灭活血清是连续稀释 96-井板中所示, 在图 4。100 TCID50/50 µL 病毒, 然后添加到每个井。在 1 h 潜伏期后, 允许血清中的抗体与病毒结合, 每个井中添加100µL 1.5 x 105细胞/毫升 MDCK-SIAT1 细胞。为了获得最佳结果, MDCK-SIAT1 的单元格应为 TCID50和锰分析的 75-95% 70-100 (图 1)。然后在37° c、5% CO 2 中孵育 18-20 h.经过一夜的孵化, 每井的病毒数量检测和量化的流感一个 NP ELISA (图 3)。
每个井中的 OD 代表在含有中和抗体的连续稀释的血清中存在的 MDCK-SIAT1 细胞中病毒感染和复制的数量。它可以绘制反对血清稀释 (图 5)。达到50% 中和的最高血清稀释的倒数被认为是血清样品的抗体效价。图 5包含2例对 A/HongKong/4801/2014 a (H3N2) 3 c. 2 a 病毒和 post-influenza 疫苗接种的配对血清测试结果的示例。与患者 1 pre-vaccination 血清, 没有血清稀释抑制病毒感染 (图 5, 浅蓝色曲线), 因此被认为是阴性 (< 10)。相比之下, 该患者的 post-vaccination 血清抑制病毒复制, 表明疫苗诱导中和抗体。这种血清的第三稀释是最高稀释低于50% 的截止, 因此, 这个病人有一个 post-vaccination 的效价为 40 (图 5, 深蓝色曲线)。相比之下, 第二个患者 pre-vaccination 的血清中含有已存在的中和抗体, 其效价为320。接种后, 滴度增加到 > 1280。在这种情况下, 在 1:10 pre-dilution 的血清, 没有抗体结束滴度达到。血清应重新在较高的 pre-dilution, 以达到最终的效价。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56448/56448fig1.jpg"> 图 1.MDCK-SIAT1 细胞培养.MDCK-SIAT1 细胞。(A) MDCK-SIAT1 汇合单层 (> 100% 70-100) 进行病毒接种;(B) 在日志阶段 MDCK-SIAT1 单元格, 其 75-95% 70-100 用于 TCID50和 MN 检测。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56448/56448fig2.jpg"> 图 2.TCID 板布局.病毒传染性由 TCID50确定。病毒的股票是 pre-diluted 到 10-2, 然后串行稀释在½日志每稀释到 10-7 (列 1-11), 在8复制每稀释 (排 H)。列12用作单元格的唯一控件。TCID50的病毒储存可以使用芦苇-明奇方法计算。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56448/56448fig3v3.jpg"> 图 3.用 MDCK-SIAT1 细胞进行锰测定的示意图.50µL 的热灭活血清是连续2倍稀释成96口井板。50µL 100 TCID50 A (H3N2) 病毒, 然后添加到每个井。板在37° c 下孵育1小时, 以允许病毒和抗体结合。然后在每个井中添加 1.5 x 104 MDCK-SIAT1 单元格。在37° c、5% CO2中孵育 18-20 h 的板。在夜间孵化后, 盘子被洗涤和固定, 每个井中的病毒数量是用流感 A NP 单克隆抗体的 ELISA 进行量化的。血清抗体滴是根据病毒控制和细胞控制所定义的切断来计算的。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56448/56448fig3v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56448/56448fig4.jpg"> 图 4.锰测定板的布局.热灭活血清是 pre-diluted 在 1:10, 然后连续2倍稀释在 96-井板 1-10。病毒控制 (仅病毒和细胞, 没有血清) 和细胞控制 (仅细胞, 没有病毒和没有血清) 在专栏12。11栏用于背部滴定或控制血清。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56448/56448fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56448/56448fig5.jpg"> 图 5.用 MDCK-SIAT1 细胞对 A/HongKong/4801/2014 A (H3N2) 病毒进行了人血清的锰测定结果.用 MDCK-SIAT1 细胞对 A/HongKong/4801/2014 A (H3N2) 病毒进行了两例 post-2016-17 和季节性流感疫苗的血清检测。中和滴是最高的血清稀释的倒数, 达到50% 病毒中和, 由箭头在每条曲线表明。病人 1: pre-vaccination 效价 < 10, post-vaccination 效价 40;病人 2: pre-vaccination 效价 320, post-vaccination 效价 > 1280。

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

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

measuring-influenza-neutralizing-antibody-responses-to-h3n2-viruses

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

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

18.0K 次观看.  Centers for Disease Control and Prevention. 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。

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

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

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

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 ...

科研

免疫与感染

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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 ...

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

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

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 ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

医学

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

磷酸钙转染法制备假型H5禽流感病毒及抗体中和活性测定

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

针对中和抗体的流感病毒逃逸变异的产生

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

一种测量流感神经氨酸酶抑制抗体滴度的测定法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...

Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

Source: Lin, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54897">Optimization of a Quantitative Micro-neutralization Assay.</a> J. Vis. Exp. (2016)
This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

使用基于图像的微量中和测定分析病毒之间的抗原关系

1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) ...