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ADCC Assay Protocol
抗体依赖的细胞介导细胞毒性(ADCC)的检测   

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Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections and cancers in vivo. Here we developed a flow cytometry based protocol for ADCC assay using human peripheral blood mononuclear cells (PBMCs) as effector cells. Using this protocol, we determined the ADCC activity of convalescent plasma IgGs from six H1N1-infected human subjects in China, and identified two dominant ADCC epitopes, designated E1 [amino acid (AA) 92-117] and E2 (AA 124-159), on haemagglutinin of pandemic H1N1 influenza virus by epitope mapping of the convalescent plasma IgGs with different levels of ADCC activity. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer a comprehensive immune protection against viral infections.

Keywords: ADCC(ADCC), Influenza Virus(流感病毒), Flowcytometry(流式细胞术), 7AAD Viability Staining(7aad活性染色), Membrane labeling(膜标签)

Materials and Reagents

  1. Raji cells (ATCC)
  2. A/California/04/2009 (H1N1) influenza virus (Reference Laboratory)
  3. Turkey red blood cells (from animal)
  4. Tissue culture medium with serum (complete medium) RPMI 1640 (Life Technologies) containing 10% heat-inactivated fetal calf serum (FCS) and 2% L-glutamine
  5. Serum, albumin, or other system-compatible protein (FBS) (Life Technologies, catalog number: 10099141 )
  6. PKH67 cell labelling dye (Sigma-Aldrich, catalog number: MIDI67 )
  7. 7-AAD Stain (Life Technologies, catalog number: A1310 )
  8. PBS (Calcium Magnesium free) (Life Technologies, catalog number: 10010-023 )
  9. RPMI 1640 media (Life Technologies, catalog number: 12633-020 )
  10. Pen/Strep (Life Technologies, catalog number: 10378016 )
  11. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )

Equipment

  1. Round bottomed 96-well plate
  2. Temperature controlled centrifuge
  3. Polypropylene conical bottom centrifuge tubes (4-15 ml)
  4. Bio-safety Cabinet
  5. Haemocytometer or cell counter
  6. Slides and coverslips
  7. Instrument (s) for fluorescence analysis (Flow cytometer)
  8. 37 °C, 5% CO2 incubator

Procedure

  1. Preperation of Target Cells
    1. Infect Raji cells at a MOI 2, 48 h before assay with A/California/04/2009 (H1N1) influenza virus in BSL-3 facility at a multiplicity to give about 80-95% infected cells. Detail procedure in Note 4.
    2. Take a sample of target cells 48 h after infection in order to assess the incidence of infected cells through its ability to produce hem-agglutination with Turkey red blood cells and binding with polyclonal antibodies. Detail procedure in Note 5.
    3. 2 x 106 cells washed twice with PBS.
    4. PKH67 cell labelling performed as per the protocol for General Cell Membrane Labelling by Sigma-Aldrich.

  2. Preparation of Effector Cells (Isolation of PBMCs)
    1. Isolate PBMCs from Blood samples of healthy volunteers, according to method given by Panda and Ravindran (2013).
    2. Wash twice isolated PBMCs with PBS.

  3. Antibody-dependent cell-mediated cytotoxicity assays (ADCC)
    Antibody-dependent cell toxicity was determined using the flow-cytometry based method employing the principle of live and dead cell discrimination (Srivastava et al., 2013).
    1. Dispense 5.0 x 104 labelled target cells in 50 μl RPMI 1640 media in each well as per the layout given bellow in round-bottomed 96 well plate in duplicate.
    2. Add 50 μl of antibodies (total IgGs from plasma of infected patients 1μg/ml and/or 5 μg/ml resulting in 0.5 μg/ml and 2.5 μg/ml) to the wells as per layout.
    3. Incubate for 15 min at 37 °C in CO2 incubator.
    4. Add unlabelled normal PBMC effector cells in 100 μl of RPMI 1640/0.5% Pen/Strep at a concentration of 2.5 x 107 cells/ml to each well as per the layout.
    5. Incubate cells for 2 h at 37 °C in CO2 incubator.
    6. After 2 h add 1 µl of the fluorescent dead cell dye 7-amino-actinomycin-D (7-AAD).
    7. Incubate at 4 °C in dark for 20 min.
    8. Analyse cells on FACS machine. A total of 5,000 target cells acquire.
    9. Determine percentage cell death by software analysis of four identifiable cell populations, live effector cells (no dye), dead effector cells (7-AAD only), live target cells (PKH-67 only) and dead target cells (PKH-67 and 7-AAD).

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      Unstained Target Cells
      Unstained Effector Cells
      PKH67 Stained Target Cells
      7AAD Stained Target Cells
      PKH67 Stained Target Cells + media + 7AAD Stain
      PKH67 Stained Target Cells + 5 µl 1% Triton X-100 + 7AAD Stain
      PKH67 Stained Target Cells + Antibody + 7AAD Stain

  4. FACS analysis step
    1. Identify total (Live and dead) target cell zone (FSC Vs SSC) (5,000) (Well no 1).
    2. Identify effector cell zone (FSC Vs SSC) (5,000) (Well no 2).
    3. Acquire PKH67 positive cells (5,000) (Well no 3).
    4. Acquire 7AAD positive cells (5,000) (Well no 4).
    5. Acquire all wells 5, 6 and 7 (5,000 PKH67 positive cells).
    6. Locate double positive population (PKH67 and 7AAD) by gating PKH67 cells and then gating 7AAD positive cells.

Notes

  1. Assay controls will be used to define cell populations included target cells without antibody (background cell death) and target cells with 5 µl 1% Triton X-100 (maximum fluorescence).
  2. Data presented as % increase in ADCC = (% cell death in presence of IgG - % cell death in absence of IgG)/(% Cell death in maximum lysis - % cell death in absence of IgG) x100.
  3. Experiments should be repeated for each IgG using pooled PBMCs from three healthy volunteers.
  4. Infection of Raji cells (briefly)
    1. 10 ml of 2 x 105 cells/ml wash 3 times with 5 ml of RPMI1640 containing 2 μg/ml of TPCK-trypsin.  
    2. Remove media and inoculate A/California/04/2009 (H1N1) influenza virus at a MOI2.  
    3. Allow inoculum to adsorb for 60 min at 37 °C.
    4. Gently wash with 6 ml of media (RPMI1640) containing 2 μg/ml of TPCK trypsin without serum.
    5. Add 5 ml of media (RPMI1640) containing 2 μg/ml of TPCK trypsin with 2% calf serum to T-25 flasks.
  5. Hem-agglutination Assay (HA)
    1. Prepare culture supernatant, serially dilute virus in PBS.
    2. Take 50 μl of each dilution and place in pre-labeled U bottom 96-well plate.
    3. Add 50 μl of a 0.5% turkey Red Blood Cells suspension to each tube and mix by agitation.
    4. Allow tubes to sit undisturbed at room temperature for 30 min.
    5. After 30 min, record results. A negative tube is seen as a perfectly outlined round "button" of cells settled at the bottom of the tube. Intermediately positive results are seen with irregular clumps of cells at the bottom of the tube. Positive results are seen with a uniform film that lacks a distinct shape covering the bottom of the tube.

Acknowledgments

We wish to thank Kwok-Yung Yuen and Kelvin KW To for providing the patient plasmas, Hong-Lin Chen and Zhiwei Chen for influenza virus H1N1 strains and the recombinant plasmid containing the full-length H1N1 HA gene, Dimiter S Dimitrov and Zhongyu Zhu for pYD7 yeast plasmid, and Martial Jaume for Raji cell line. We thank Kwok-Yung Yuen, Kelvin KW To, Linqi Zhang, Qi Zhao, Li Liu and Jia Guo for helpful discussions, and Yanyu Zhang and Jingjing Li for technical assistance. This work was supported by China 12th 5-year Mega project (# 2012ZX10001006) and Small Project Funding (# 201109176176 and # 201007176258) from the University of Hong Kong to M-Y. Z.

References

  1. Panda, S. and Ravindran, B. (2013). Isolation of Human PBMCs. Bio-protocol 3(3): e323. 
  2. Srivastava, V., Yang, Z., Hung, I. F., Xu, J., Zheng, B. and Zhang, M. Y. (2013). Identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic H1N1 influenza virus. J Virol 87(10): 5831-5840. 

简介

抗体依赖性细胞介导的细胞毒性(ADCC)桥接先天和适应性免疫,并且其涉及体液和细胞免疫应答。已经发现ADCC是针对体内病毒感染和癌症的免疫保护的主要途径。在这里,我们开发了基于流式细胞术的协议,使用人类外周血单核细胞(PBMCs)作为效应细胞ADCC测定。使用这个协议,我们确定在中国六个H1N1感染人类受试者的恢复期血浆IgG的ADCC活性,并确定两个显性ADCC表位,指定E1 [氨基酸(AA)92-117]和E2(AA 124-159) ,通过具有不同水平的ADCC活性的恢复血浆IgG的表位作图对大流行性H1N1流感病毒的血凝素进行。我们的研究可能有助于设计可以引发具有高ADCC活性的抗体的免疫原。设计为包括有效的广泛中和抗体和ADCC表位的结构决定簇的疫苗免疫原可以赋予针对病毒感染的全面免疫保护。

关键字:ADCC, 流感病毒, 流式细胞术, 7aad活性染色, 膜标签

材料和试剂

  1. Raji细胞(ATCC)
  2. A/California/04/2009(H1N1)流感病毒(参考实验室)
  3. 土耳其红细胞(来自动物)
  4. 含有10%热灭活胎牛血清(FCS)和2%L-谷氨酰胺的血清(完全培养基)RPMI 1640(Life Technologies)的组织培养基。
  5. 血清,白蛋白或其他系统相容性蛋白(FBS)(Life Technologies,目录号:10099141)
  6. PKH67细胞标记染料(Sigma-Aldrich,目录号:MIDI67)
  7. 7-AAD Stain(Life Technologies,目录号:A1310)
  8. PBS(不含钙镁)(Life Technologies,目录号:10010-023)
  9. RPMI 1640培养基(Life Technologies,目录号:12633-020)
  10. Pen/Strep(Life Technologies,目录号:10378016)
  11. Triton X-100(Sigma-Aldrich,目录号:T8787)

设备

  1. 圆底96孔板
  2. 温度控制离心机
  3. 聚丙烯锥形底部离心管(4-15 ml)
  4. 生物安全柜
  5. 血细胞计数器或细胞计数器
  6. 幻灯片和盖玻片
  7. 用于荧光分析的仪器(流式细胞仪)
  8. 37℃,5%CO 2培养箱

程序

  1. 靶细胞的预处理
    1. 在用A/California/04/2009(H1N1)流感病毒在BSL-3设备中测定之前,在MOI 2,4,8小时感染Raji细胞,以多重性给予约80-95%的感染细胞。 注释4中的详细过程。
    2. 感染后48小时取目标细胞样本,以评估感染细胞的发生率,通过其与土耳其红细胞产生hem凝集和与多克隆抗体结合的能力。 注释5中的详细过程。
    3. 2×10 6个细胞用PBS洗涤两次
    4. PKH67细胞标记根据Sigma-Aldrich的通用细胞膜标记方案进行。

  2. 效应细胞的制备(PBMC的分离)
    1. 根据Panda和Ravindran(2013)给出的方法,从健康志愿者的血液样品中分离PBMC。
    2. 用PBS洗涤两次分离的PBMC。

  3. 抗体依赖性细胞介导的细胞毒性测定(ADCC)
    使用基于活细胞和死细胞鉴别原理的基于流式细胞术的方法测定抗体依赖性细胞毒性(Srivastava等人,2013)。
    1. 按照在圆底96孔板中给出的布局一式两份,在每个孔中在50μlRPMI 1640培养基中分配5.0×10 4个标记的靶细胞。
    2. 按照布局向孔中加入50μl抗体(来自感染患者的血浆的总IgG,1μg/ml和/或5μg/ml,得到0.5μg/ml和2.5μg/ml)。
    3. 在37℃下在CO 2培养箱中孵育15分钟
    4. 按照布局将未标记的正常PBMC效应细胞以2.5×10 7个细胞/ml的浓度加入到100μl的RPMI 1640/0.5%Pen/Strep中。
    5. 在37℃下在CO 2培养箱中孵育细胞2小时
    6. 2小时后加入1μl荧光死细胞染料7-氨基 - 放线菌素D(7-AAD)。
    7. 在4℃避光孵育20分钟。
    8. 在FACS机上分析细胞。 总共有5,000个靶细胞获得
    9. 通过软件分析四个可识别的细胞群,活效应细胞(无染料),死效应细胞(仅7-AAD),活靶细胞(仅PKH-67)和死靶细胞(PKH-67和7 -AAD)。

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      未染色的目标单元格
      未染色效应细胞
      PKH67染色靶细胞
      7AAD染色靶细胞
      PKH67染色靶细胞+培养基+ 7AAD染色
      PKH67染色的靶细胞+5μl1%Triton X-100 + 7AAD染色
      PKH67染色的靶细胞+抗体+ 7AAD染色

  4. FACS分析步骤
    1. 识别总(活和死)靶细胞区(FSC vs SSC)(5,000)(孔1)
    2. 识别效应细胞区(FSC vs SSC)(5,000)(2号孔)
    3. 获得PKH67阳性细胞(5,000)(孔3)
    4. 获得7AAD阳性细胞(5,000)(孔4)
    5. 获得所有孔5,6和7(5,000个PKH67阳性细胞)
    6. 通过门控PKH67细胞,然后选通7AAD阳性细胞定位双阳性群体(PKH67和7AAD)。

笔记

  1. 测定对照将用于定义包含无抗体的靶细胞(背景细胞死亡)的细胞群和具有5μl1%Triton X-100(最大荧光)的靶细胞。
  2. 数据表示为ADCC%增加%(IgG存在时的细胞死亡% - IgG不存在时的细胞死亡%)/(最大裂解中的细胞死亡% - 不存在IgG时的细胞死亡%)x100。
  3. 应使用来自三个健康志愿者的汇集的PBMC对每个IgG重复实验
  4. 感染Raji细胞(简要)
    1. 10ml 2×10 5个细胞/ml,用5ml含有2μg/ml TPCK-胰蛋白酶的RPMI1640洗涤3次。  
    2. 除去培养基并在MOI2接种A/California/04/2009(H1N1)流感病毒。  
    3. 允许接种物在37℃吸附60分钟。
    4. 用6ml含有2μg/ml无血清的TPCK胰蛋白酶的培养基(RPMI1640)轻轻洗涤。
    5. 将含有2μg/ml含2%小牛血清的TPCK胰蛋白酶的5ml培养基(RPMI1640)加入T-25烧瓶中。
  5. 血凝集测定(HA)
    1. 准备培养上清,在PBS中连续稀释病毒。
    2. 取50μl的每个稀释,并置于预标记的U底96孔板。
    3. 向每个管中加入50μl的0.5%火鸡红细胞悬浮液,并通过搅拌混合
    4. 允许管在室温下静置30分钟。
    5. 30分钟后,记录结果。 阴性管被视为位于管底部的细胞的完全轮廓的"按钮"。 在管底部观察到不规则的细胞团的中间阳性结果。 使用均匀的薄膜看到正面的结果,其缺乏覆盖管底部的明显形状

致谢

我们要感谢Kwok-Yung Yuen和Kelvin KW To为流感病毒H1N1毒株提供患者血浆,Hong-Lin Chen和Zhiwei Chen,以及含有全长H1N1 HA基因的重组质粒Dimiter S Dimitrov和Zhongyu Zhu pYD7酵母质粒和用于Raji细胞系的Martial Jaume。我们感谢Kwok-Yung Yuen,Kelvin KW To,Linqi Zhang,Qi Zhao,Li Liu和Jia Guo进行了有益的讨论,并与Yanyu Zhang和Jingjing Li取得了技术援助。这项工作得到了中国第12个5年大型项目(#2012ZX10001006)和从香港大学到M-Y的小项目资金(#201109176176和#201007176258)的支持。 Z.

参考文献

  1. Panda,S。和Ravindran,B。(2013)。 人PBMC的分离。生物协议 3(3):e323 。
  2. Srivastava,V.,Yang,Z.,Hung,I.F.,Xu,J.,Zheng,B.and Zhang,M.Y。 在大流行性H1N1流感病毒的血凝素抗原上鉴定显性抗体依赖性细胞介导的细胞毒性表位。 J Virol 87(10):5831-5840。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Srivastava, V., Yang, Z., Hung, I. F., Xu, J., Zheng, B. and Zhang, M. (2014). ADCC Assay Protocol. Bio-protocol 4(2): e1029. DOI: 10.21769/BioProtoc.1029.
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