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Hemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies
单克隆抗体介导的流感病毒血细胞凝集抑制(HI)试验   

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Abstract

Heamagglutination is inhibited when antibodies are present because antibodies to the influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. Human monoclonal antibodies generated from single human B cells were tested to characterize their ability to inhibit hemagglutination against virus A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2).

Keywords: Haemagglutination inhibition(血凝抑制), Influenza(流感), Antibody(抗体)

Materials and Reagents

  1. Materials
    1. 96 well microtiter plates (V bottom) (Corning, catalog number: 3897 )
    2. Tips for multichannel pipette (Gilson, model: D10 , D200 and D1000 )
    3. Centrifuge tubes (SARSTEDT AG & Co, catalog number: 72.690 )

  2. Reagents
    1. Viral antigen
      1. H1N1
        A/California/07/2009
      2. H3N2
        A/Brisbane/10/2007
        Note: Viruses were amplified in embryonated eggs. Refer to Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza, WHO.
    2. Erythrocytes
      Turkey red blood cells (RBCs)
      Note: Both in-house RBCs and commercial RBCs are usable.
    3. Antibody
      Human monoclonal antibodies CT146, CT147, CT149, CT164 and CT166
      Notes:
      1. Maximum concentration of antibody is 20 μg/ml.
      2. Expressed in CHO cells.
      3. Purified according to manufacturer’s instruction [HiTrapTM MabSelect SuRe (GE Healthcare, catalog number: 11-0034-93 )].
    4. Other reagents
      1. Receptor destroying enzyme (RDE) (DENKA SEIKEN CO., catalog number: 370013 )
      2. Phosphate buffered saline (1x PBS) (pH 7.2) (Sigma-Aldrich, catalog number: P4417 )
      3. Physiological saline (0.85% NaCl) (Sigma-Aldrich, catalog number: S9888 )

Equipment

  1. Hemocytometer (Hausser Scientific, catalog number: 1492 )
  2. Multichannel pipette (Gilson, model: PIPETMAN Neo® Multichannel )
  3. Centrifuge (Beckman Coulter, model: Allegra X-15R )
  4. Water bath (JULABO GmbH, model: MB13 )
  5. Inverted routine microscopy (Nikon Instruments Inc., model: TS-100 )

Procedure

  1. Preparation of erythrocytes
    1. Prepare the diluted RBC for washing step: 5 ml of RBC + 45 ml of 1x PBS (room temperature).
    2. Washing step (just one time): Centrifuge the diluted RBC, 2,000 x g, 10 min, 25 °C.
    3. Discard supernatant and then suspend the RBC pellet with 20 ml (initial volume) of 1x PBS.
    4. 1:100 dilution (0.5 ml of RBC suspension from step A3 + 49.5 ml of 1x PBS).
    5. Transfer 10 μl of RBC from step d onto a hemocytometer and count the RBC in each of the 4 squares to calculate the final volume of RBC.
    6. Calculate the final volume of RBC by formula below (Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza, WHO).
      1. Formula
        Final volume = total number of cells counted X initial volume /160
      2. Example of calculation
        Initial volume =20
        Total number of RBC = 320
        Final volume = 320 x 20/160 (160 is used as constant when use avian red blood cells; Chicken or turkey) = 40
        Then, add 20 ml of 1x PBS to 20 ml of RBC to make 40 ml and use it for HI assay.

  2. Hemagglutination titration of viral antigen (Serial dilution is described in Figure 1)
    1. Add 50 μl of PBS to the V bottom 96 wells in column 2 to 12.
    2. Add 100 μl of each antigen to the first wells of rows.
    3. Make serial 2-fold dilutions by transferring 50 μl from the first to successive wells of each row using multi-channel pipette.
    4. Discard the final 50 μl.
    5. Add 50 μl of prepared RBCs to each well.
    6. Incubate the plate at room temperature for 30 min to 1 h.
    7. Record the HAU results (refer to Example of HAU titers as shown in Figure 2).


      Figure 1. Serial dilution of test sample for HAU titration. Add 50 μl of PBS to all wells in column 2 to 12 of a v-bottom 96-well plate and then add 100 μl of test sample to the first well of each row. Transfer 50 μl of sample to the next wells using a multi-channel pipette to make a 2-fold dilution. Dilution factor is 1 to 2,048.


      Figure 2. Example of HAU titers. Hemagglutinin on the surface of influenza virus binds to the sialic acid receptors of red blood cells and creates a lattice structure. The agglutinated lattice maintains the red blood cells in a suspended and shows as a reddish solution.  HAU titer is the dilution factor of the last well showing reddish solution. HAU titer of sample 1 to sample 8 is 16, 8, 32, 32, 32, 16, 32 and 64.

  3. Preparation of standardized antigen for HI test
    1. Prepare 4 hemagglutination units (HAU) of antigen by dilution of antigen.
      Example of dilution: If antigen HAU is 16, 4 fold dilution with 1x PBS for 4 HAU.

  4. Hemagglutination inhibition (HI) test (Serial dilution is described in Figure 3)
    1. Add 25 μl of PBS to the V bottom 96 wells in column 2 to 12.
    2. Add 50 μl of the antibody (diluted with PBS, 1:10) to the well in row A1 to H1.
    3. Transfer 25 μl of the antibody from column 1 to 12 for 2 fold serial dilution.
    4. Add 25 μl of standardized antigen to all wells of plates.
    5. Mix the plates using a laboratory shaker for 10 sec or by manually agitating the plates thoroughly.
    6. Cover the plates and incubate at room temperature for 30 min.
    7. Add 50 μl of prepared RBC to all wells and incubate at room temperature for 30 min.
    8. Observe the hemagglutination inhibition. If sample inhibits hemagglutination, RBCs appear as a dot by sink to bottom of well. Record the results by refering to Example of HI titers as shown in Figure 4.


      Figure 3. Serial dilution of test sample for HI assay. Add 25 μl of PBS to all wells in column 2 to 12 of a V-bottom 96-well plate and then add 50 μl of test sample (pre diluted 1:10) to the first well of each row. Transfer 25 μl of sample to the next wells using a multi-channel pipette to make a 2-fold dilution. Dilution factor is 10 to 20,480.


      Figure 4. Example of HI titers. If antibodies bind to the viral particles, the influenza virus is effectively blocked from causing hemagglutination. The HI titer value is the last dilution factor of antibody showing completely inhibited hemagglutination. HI titer of sample 1 to sample 8 is 160, 80, 80, 320, 80, 160, 80 and 80.

Notes

  1. Inactivation of nonspecific inhibitors (in case of using serum)
    1. Reconstitute the RDE with the volume of physiological saline (0.85% NaCl).
    2. Add 3 volumes of RDE to 1 volume of antibody.
    3. Incubate overnight in 37 °C water bath.
    4. Heat in a 56 °C water bath for 30 min to inactivate any remaining RDE.
    5. Cool at room temperature and add 6 volumes of physiological saline. The final dilution of antibody is 1:10.

Acknowledgments

We thank Dr. Jun Myung Kim and Dr. Sang Hoon Han for providing blood samples from Severance Hospital, and Dr. Yasuo Watanabe of SC World, Inc. for assistance in ISAAC assays for antibody screening. This study was supported by a grant of the Korea Healthcare technology R&D Project‚ Ministry of Health&Welfare‚ Republic of Korea. (Grant No. A103001), the National Natural Science Foundation of China (NSFC, Grant No. 81401671), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB08020100), the China National Grand S&T Special Project (Grant No. 2015ZX09304005) and. G. F. G. is a leading principal investigator of the NSFC Innovative Research Group (Grant No. 81321063). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

References

  1. Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza. WHO global influenza surveillance network...

简介

当存在抗体时,凝血抑制被抑制,因为针对流感病毒的抗体将阻止病毒附着于红细胞。 防止血细胞凝集的抗体的最高稀释度称为HI滴度。 测试从单个人B细胞产生的人单克隆抗体,以表征其抑制关于病毒A/California/07/2009(H1N1)和A/Brisbane/10/2007(H3N2)的凝血的能力。

关键字:血凝抑制, 流感, 抗体

材料和试剂

  1. 材料
    1. 96孔微量滴定板(V底)(Corning,目录号:3897)
    2.多通道移液器(Gilson,型号:D10,D200和D1000)的提示
    3.离心管(SARSTEDT AG& Co,目录号:72.690)

  2. 试剂
    1. 病毒抗原
      1. 一个。 H1N1
        A/California/07/2009
      2. H3N2
        A/Brisbane/10/2007
        注意:在含胚卵中扩增病毒。 请参阅手册 实验室诊断和流感病毒学监测,WHO。
    2. 红细胞
      土耳其红细胞(RBC)
      注意:内部RBC和商业RBC都可以使用。
    3. 抗体
      人单克隆抗体CT146,CT147,CT149,CT164和CD166
      注意:
      1. 抗体的最大浓度为20μg/ml
      2. 在CHO细胞中表达
      3. 根据制造商的说明纯化[HiTrap TM MabSelect SuRe(GE Healthcare,目录号:11-0034-93)]
    4. 其他试剂
      1. 受体破坏酶(RDE)(DENKA SEIKEN CO。,目录号:370013)
      2. 磷酸盐缓冲盐水(1x PBS)(pH 7.2)(Sigma-Aldrich,目录号P4417)
      3. 生理盐水(0.85%NaCl)(Sigma-Aldrich,目录号:S9888)

设备

  1. 血细胞计数器(Hausser Scientific,目录号:1492)
  2. 多通道移液器(Gilson,型号:PIPETMAN Neo 多通道)
  3. 离心机(Beckman Coulter,型号:Allegra X-15R)
  4. 水浴(JULABO GmbH,型号:MB13)
  5. 反向常规显微镜(Nikon Instruments Inc.,型号:TS-100)

程序

  1. 红细胞的制备
    1. 准备稀释的RBC进行洗涤步骤:5ml RBC + 45ml 1×PBS(室温)
    2. 洗涤步骤(仅一次):将稀释的RBC,2,000×g/g,10分钟,25℃离心。
    3. 弃去上清液,然后用20ml(初始体积)1x PBS悬浮RBS沉淀
    4. 1:100稀释液(0.5ml来自步骤A3的RBC悬浮液+49.5ml1×PBS)
    5. 转移到10微升的RBC的步骤d血细胞计数器和计数 RBC在4个方格中的每一个中计算RBC的最终体积
    6. 通过以下公式计算RBC的最终体积(公式为 参考手册实验室诊断和病毒学 流感监测,世卫组织)。
      1. 公式为
        最终体积=计数的细胞总数×初始体积/160
      2. 计算示例
        初始音量= 20
        RBC总数= 320
        最终体积= 320 x 20/160(使用禽红血球时为160;鸡或火鸡为常数)= 40
        然后,向20ml RBC中加入20ml 1×PBS,制成40ml,用于HI测定。

  2. 病毒抗原的凝血(HAU)滴定(系列稀释在图1中描述)
    1. 加入50μlPBS到第2列到第12列的V底96个孔。
    2. 将100微升的每种抗原添加到行的第一口井。
    3. 通过转移50μl从第一次进行连续2倍稀释   每行的连续孔使用多通道移液器
    4. 弃去最终50μl。
    5. 向每个孔中加入50μl制备的RBC
    6. 将板在室温下孵育30分钟至1小时
    7. 记录HAU结果参考图2下面的HAU滴度的实施例

      图1. HAU滴定的测试样品的系列稀释液。 添加50μl 的PBS加入v-底96孔板的第2-12列的所有孔中 然后向每行的第一个孔中加入100μl测试样品。 使用多通道移液器将50μl样品转移到下一个孔 以进行2倍稀释。稀释因子为1至2,048

      图2.HAU滴度的实施例。表面的血凝素 流感病毒结合红细胞的唾液酸受体 然后创建晶格结构。凝集的晶格保持 红细胞悬浮并显示为微红色溶液。 凝集效价(HAU)是最后一个孔的稀释因子 显示带红色的解决方案。样品1至样品8的HAU滴度为 16,8,32,32,32,16,32及64条。

  3. HI测试的标准抗原制备
    1. 通过稀释抗原制备抗原的4个凝血单位(HAU) 稀释实施例:如果抗原HAU为16,4倍HAU,用1x PBS稀释4倍。

  4. 凝集抑制(HI)试验(系列稀释在图3中描述)
    1. 将25μlPBS加入第2至12列的V底96孔中。
    2. 加入50μl抗体(用PBS稀释,1:10)到A1行到H1行的孔
    3. 将25μl抗体从柱1转移至12,进行2倍连续稀释
    4. 向板的所有孔中加入25μl标准抗原
    5. 使用实验室振动器混合板10秒或通过手动完全搅拌板
    6. 盖上板,在室温下孵育30分钟。
    7. 向所有孔中加入50μl制备的RBC,并在室温下孵育30分钟
    8. 观察血凝抑制。如果样品抑制 血细胞凝集,RBCs通过下沉到孔底部显示为点。 记录结果参考下面HI滴度的实施例(图4)

      图3.用于HI测定的测试样品的系列稀释 。向V-底96孔的第2-12列的所有孔中加入25μlPBS 然后加入50μl测试样品(预稀释1:10)至第一个 井的每行。使用多转移25μl样品到下一口井  通道移液器使2倍稀释。稀释因子为10至 20,480。


      图4. HI滴度的实施例。如果抗体结合  到病毒颗粒,流感病毒被有效阻止 引起血细胞凝集。 HI滴度值是最后一个稀释因子  的抗体显示完全抑制的血凝。 HI滴度 样品1至样品8为160,80,80,320,80,160,80和80。

笔记

  1. 非特异性抑制剂的失活(在使用血清的情况下)
    1. 用生理盐水(0.85%NaCl)体积重建RDE
    2. 将3倍体积的RDE加入1倍体积的抗体中
    3. 在37℃水浴中孵育过夜
    4. 在56℃水浴中加热30分钟以灭活任何剩余的RDE
    5. 在室温下冷却并加入6体积的生理盐水。抗体的最终稀释度为1:10。

致谢

我们感谢Jun Myung Kim博士和Sang Hoon Han博士提供Severance医院的血液样本,SC World公司的Yasuo Watanabe博士为抗体筛选的ISAAC分析提供帮助。本研究得到韩国卫生和福利部韩国医疗保健技术研究与发展项目的资助。 (国家自然科学基金资助项目(编号A103001),国家自然科学基金(NSFC,批准号81401671),中国科学院战略重点研究计划(编号XDB08020100),中国国家大科技(批准号2015ZX09304005)和。 G.F.G.是NSFC创新研究组的首席主要研究者(批准号81321063)。本报告的结果和结论是作者的结果和结论,不一定代表疾病控制和预防中心或有毒物质和疾病登记处机构的观点。

参考文献

  1. 实验室诊断和流感病毒学监测手册世卫组织全球流感监测网络 。。 。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wu, Y., Cho, M., Shore, D., Song, M., Choi, J., Jiang, T., Deng, Y., Bourgeois, M., Almli, L., Yang, H., Chen, L., Shi, Y., Qi, J., Li, A., Yi, K. S., Chang, M., Bae, J. S., Lee, H., Shin, J., Stevens, J., Hong, S., Qin, C., Gao, G. F., Chang, S. J. and Donis, R. O. (2016). Hemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies. Bio-protocol 6(11): e1828. DOI: 10.21769/BioProtoc.1828.
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