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Immunoplaque Assay (Influenza Virus)
免疫斑块试验(流感病毒)   

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Abstract

Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, genetic stability and purity of the virus population.

Keywords: Influenza(禽流感), Plaque Assay(试验板), Quantification(量化的)

Materials and Reagents

  1. MDCK cells
    Please note that there are multiple variants of MDCK on American Type Culture Collection (ATCC). This protocol has been tested for MDCK (CCL-34), MDCK.1 (CRL-2935) and MDCK.2 (CRL-2936)
  2. Recombinant A/California/04/2009 virus produced from 12 plasmids reverse genetic system (Generous gift from Professor Toru Takimoto from University of Rochester, New York, USA) (Bussey et al., 2010)
  3. Trypsin/EDTA (Mediatech, Cellgro®, catalog number: 25-053-CI )
  4. SeaPlaque Agarose (Lonza, catalog number: 50100 )
  5. TPCK-Trypsin (Thermo Fisher Scientific, catalog number: 20233 )
  6. PBS without Ca2+ and Mg2+ (Mediatech, Cellgro®, catalog number: 21-030-CV )
  7. DPBS with Ca2+ and Mg2+ (Mediatech, Cellgro®, catalog number: 21-040-CV )
  8. 10% formalin (Sigma-Aldrich, catalog number: HT501128-4L )
  9. TritonX-100 (Sigma-Aldrich, catalog number: T9284 )
  10. 5% goat serum in DPBS (Gibco, catalog number: 16210-072 )
  11. Mouse anti-NP antibody from hybridoma H16-L10-4R5 (ATCC, catalog number: HB-65 )
  12. Alkaline phosphatase conjugated Donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, catalog number: 715-056-150 )
  13. Alexa Fluor 488 conjugated Goat anti-mouse antibody (Invitrogen, catalog number: A11001 )
  14. BCIP/NBT substrate (Vector Laboratories, catalog number: SK-5400 )
  15. DMEM (Mediatech, Cellgro®, catalog number: 10-017-CV )
  16. DMEM powder (Mediatech, Cellgro®, catalog number: 50-003-PB )
  17. Heat-inactivated fetal bovine serum (Mediatech, Cellgro®, catalog number: 26140079 )
  18. HEPES (Mediatech, Cellgro®, catalog number: 25-060-CI )
  19. Penicillin/Streptomycin (Mediatech, Cellgro®, 30-002-CI )
  20. 7.5% BSA Fraction V (Gibco, catalog number: 15260 )
  21. RPMI 1640 powder (Mediatech, Cellgro®, catalog number: 50-020-PB )
  22. Tris base (US Biological, catalog number: T8600 )
  23. Sodium chloride (NaCl)
  24. Magnesium chloride (MgCl2)
  25. Sodium hydroxide (NaOH)
  26. Complete DMEM (cDMEM) (see Recipes)
  27. 2x DMEM (see Recipes)
  28. RPMI (see Recipes)
  29. 0.5% Triton X-100 in DPBS (see Recipes)
  30. Alkaline phosphatase (AP) reaction buffer (see Recipes)

Equipment

  1. Standard tissue culture equipment
  2. 12-well tissue culture plate
  3. 37 °C, 5% CO2 cell culture incubator
  4. Inverted fluorescence microscope (Optional)
  5. Rocker
  6. Laboratory spatula
  7. Heated stir plate
  8. Vortex
  9. Axiovert 200 microscope

Software

  1. IPlab3.6.5 software

Procedure

  Day 1

  1. Each well on a 12-well plate is seeded with 5 x 105 MDCK cells suspended in 1 ml of cDMEM.
  2. The plate is incubated in the 5% CO2 incubator at 37 °C overnight.

  Day 2 (or until cells reach 95% - 100% confluence)

  1. Viruses are serially diluted (10-fold) in RPMI, vortex each dilutions and keep on ice before use.
  2. MDCK cells are rinsed with 1 ml of DPBS twice.
  3. MDCK cells are inoculated with 500 μl of viruses prepared from step 3.
  4. The plate is incubated in 5% CO2 incubator at 37 °C for 45 min.
  5. Prepare SeaPlaque DMEM (pDMEM)
    1. 2x DMEM is warmed at 37 °C water bath and 2% SeqPlaque agarose is melted on a hot plate at about 45–50 °C with stirring (*The cap should be slightly released to prevent pressure buildup).
    2. To obtain pDMEM, pre-warm 2x DMEM and 2% agarose are mixed in 1:1 ratio in a sterile container and sit at room temperature for 5–10 min to cool down.
    3. TPCK-trypsin (final concentration: 1 μg/ml) is added to the pDMEM.
  6. Viral inoculums are aspirated and MDCK cells are rinsed with 1 ml of DPBS once.
  7. 1.5 ml pDMEM + TPCK-trypsin from step 7-c is added to each well.
  8. The plate is sat at room temperature (RT) for 10–15 min until pDMEM solidify.
  9. The plate is incubated at 5% CO2 at 37 °C in the invert orientation (lid at the bottom) for 3 days.

 Day 5 (or until visualization of plaque)

  1. MDCK cells are fixed by adding 1.5 ml of 10% formalin directly on top of the agarose and incubated at RT for 2 h.
  2. Extra formalin is aspirated and agarose from the well is removed and trashed carefully without disturbing the monolayer of cell using a laboratory spatula or equivalent tools.
    Note: Experiment can be stopped after this step by adding PBS to cover the cells and store at 4 °C.
  3. MDCK cells are permeabilized by 300 μl of 0.5% Triton-X100 in DPBS for 5 min on bench without agitation.
  4. Cells are rinsed with 1 ml of PBS twice.
  5. Cells are blocked by 300 μl of 5% goat serum in DPBS for 30 min at RT with agitation on rocker.
  6. Cells are incubated with H16-L10-4R5 antibody (1:40) in 300 μl of 5% goat serum for 1 h at RT with agitation on a rocker.
  7. Cells are rinsed with 1 ml of PBS twice.
  8. Cells are incubated with alkaline phosphatase conjugated donkey anti-mouse (1:1,000) in 300 μl of 5% goat serum for 45 min at RT with agitation on a rocker.
  9. Cells are rinsed with 1 ml of PBS twice and AP reaction buffer once.
  10. Prepare the BCIP/NBT solution.
    1. Added 2 drops of solution A, B and C into every 5 ml of AP reaction buffer.
    2. Mix by inverting the tube for 4–6 times.
  11. 300 μl of BCIP/NBT solution is added to each well. The plate is put on a rocker in dark for 30 min or until the development of color.
  12. The reaction is stopped by rinsing the plate with ddH2O.
  13. The plate is air-dried at RT.
  14. Count the number of plaques, check the plaque morphology and measure the size of plaques.

    Alternative visualization method by fluorescence, fluorescent focus assay (FFA)
    (Continuous from step 18)
    • 19.1
    • Cells are incubated with goat anti-mouse Alexa Fluor 488 antibody (1:1,000) in 300 μl of 5% goat serum for 45 min at RT in dark.
    20.1 Cells are rinsed with 1 ml of PBS twice.
    21.1 Cells are covered with 500 μl of DPBS and store at 4 °C.
    • 22.1
    • Check the plaque morphology and measure the size of plaques using an inverted fluorescence microscope (Figure 1).
      Note: The protocol is optimized for influenza viruses. Modifications may be required for use in other viruses. For instance, factors required for multiple rounds of replication of the virus should be added in the pDMEM.


      Figure 1. Diameter of Individual Plaque of A/California/04/2009/H1N1 Recombinatant Viruses (rCA0409). A. Quantification of the diameter (μm) of 100 individual plaques of two different CA0409 viruses, wild type (WT) and HA-S328Y mutants (HA328Y). Images are taken by Axiovert 200 microscope using a 5x objective and diameters of each plaque are measured by IPlab3.6.5 software. Statistics are done by one-tail Student’s t-test. B. Representative image of plaques of CA0409 viruses stained with Alexa Fluor 488, scale bar = 200 μm. Images are taken by Sensicam using IPlab 3.6.5 (Data not published) (Tse et al., 2013).

Recipes

  1. cDMEM (filtered in 0.2 μm filtration unit)
    DMEM
    432.5 ml
    Heat-inactivated fetal bovine serum         
    50 ml
    1 M HEPES                                              
    12.5 ml
    Penicillin/Streptomycin
    5 ml
  2. 2x DMEM (filtered in 0.2 μm filtration unit)
    DMEM powder
    13.48 g
    7.5% BSA Fraction V
    26.7 ml
    1 M HEPES
    12.5 ml
    Penicillin/Streptomycin
    5 ml
    ddH2O to 500 ml
  3. RPMI (filtered in 0.2 μm filtration unit)
    RPMI 1640 powder
    5.2 g
    7.5% BSA fraction V
    13.3 ml
    1 M HEPES
    12.5 ml
    Penicillin/Streptomycin
    5 ml
    ddH2O to 500 ml
  4. 0.5% Triton X-100 in DPBS
    Triton X- 100
    1 ml
    DPBS
    199 ml
  5. AP reaction buffer
    Tris
    100 mM
    NaCl 
    100 mM
    MgCl2
    5 mM
    Adjust pH to 9.5 using NaOH

Acknowledgments

We thank Jean K. Millet, Tamar Friling, Alice M. Hamilton and all of the members of the Whittaker lab for helpful discussions. We also thank the Collins lab for helpful suggestions made throughout the study.

References

  1. Bussey, K. A., Bousse, T. L., Desmet, E. A., Kim, B. and Takimoto, T. (2010). PB2 residue 271 plays a key role in enhanced polymerase activity of Influenza A viruses in mammalian host cells. J Virol 84(9): 4395-4406.
  2. Tse, L. V., Marcano, V. C., Huang, W., Pocwierz, M. S. and Whittaker, G. R. (2013). Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase. J Virol 87(9): 5161-5169.   

简介

尽管发展很久以前,斑块测定仍然是现代病毒学病毒滴度量化的黄金标准。 标准的基于结晶紫的斑块测定依赖于病毒诱导细胞病变效应(CPE)的能力,其限制了对裂解病毒的测定。 替代性病毒定量测定如50%组织培养感染测定(TCID 50)和通过Q-PCR的遗传物质定量提供了具有其自身缺点的病毒定量的不同方式。 在这里,我们修改荧光焦点测定和开发基于抗体的免疫斑检测提供可靠和可重复的病毒定量独立于CPE。 我们的测定不仅允许病毒滴度的精确测定,而且提供关于病毒动力学,遗传稳定性和病毒种群的纯度的信息。

关键字:禽流感, 试验板, 量化的

材料和试剂

  1. MDCK单元格
    请注意,在美国典型培养物保藏中心(ATCC)上有多种MDCK变体。 此协议已针对MDCK(CCL-34),MDCK.1(CRL-2935)和MDCK.2(CRL-2936)进行测试
  2. 由12个质粒反向遗传系统产生的重组A/California/04/2009病毒(来自美国纽约罗切斯特大学的Toru Takimoto教授的慷慨礼物)(Bussey等人,2010)
  3. 胰蛋白酶/EDTA(Mediatech,Cellgro ,目录号:25-053-CI)
  4. SeaPlaque Agarose(Lonza,目录号:50100)
  5. TPCK-胰蛋白酶(Thermo Fisher Scientific,目录号:20233)
  6. 没有Ca 2+和Mg 2+的PBS(Mediatech,Cellgro ,目录号:21-030-CV)的
  7. 具有Ca 2+和Mg 2+的缓冲液(Mediatech,Cellgro ,目录号:21-040-CV)的
  8. 10%福尔马林(Sigma-Aldrich,目录号:HT501128-4L)
  9. TritonX-100(Sigma-Aldrich,目录号:T9284)
  10. 5%山羊血清的DPBS(Gibco,目录号:16210-072)中
  11. 来自杂交瘤H16-L10-4R5(ATCC,目录号:HB-65)的小鼠抗NP抗体
  12. 碱性磷酸酶缀合的驴抗小鼠抗体(Jackson ImmunoResearch Laboratories,目录号:715-056-150)
  13. Alexa Fluor 488共轭的山羊抗小鼠抗体(Invitrogen,目录号:A11001)
  14. BCIP/NBT底物(Vector Laboratories,目录号:SK-5400)
  15. DMEM(Mediatech,Cellgro ,目录号:10-017-CV)
  16. DMEM粉末(Mediatech,Cellgro ,目录号:50-003-PB)
  17. 热灭活的胎牛血清(Mediatech,Cellgro ,目录号:26140079)
  18. HEPES(Mediatech,Cellgro ,目录号:25-060-CI)
  19. 青霉素/链霉素(Mediatech,Cellgro ,30-002-CI)
  20. 7.5%BSA Fraction V(Gibco,目录号:15260)
  21. RPMI 1640粉末(Mediatech,Cellgro ,目录号:50-020-PB)
  22. Tris碱(US Biological,目录号:T8600)
  23. 氯化钠(NaCl)
  24. 氯化镁(MgCl 2)
  25. 氢氧化钠(NaOH)
  26. 完成DMEM(cDMEM)(参见配方)
  27. 2x DMEM(参见配方)
  28. RPMI(见配方)
  29. 0.5%Triton X-100(参见配方)
  30. 碱性磷酸酶(AP)反应缓冲液(参见配方)

设备

  1. 标准组织培养设备
  2. 12孔组织培养板
  3. 37℃,5%CO 2细胞培养箱中培养
  4. 倒置荧光显微镜(可选)
  5. 摇杆
  6. 实验室铲
  7. 加热搅拌板
  8. 涡流
  9. Axiovert 200显微镜

软件

  1. IPlab3.6.5软件

程序

  第1天

  1. 在12孔板上的每个孔中接种悬浮在1ml cDMEM中的5×10 5个MDCK细胞。
  2. 将板在5%CO 2培养箱中在37℃温育过夜

  第2天(或直到细胞达到95%-100%汇合)

  1. 病毒在RPMI中连续稀释(10倍),涡旋每个稀释物并在使用前保持在冰上。
  2. 将MDCK细胞用1ml DPBS漂洗两次
  3. MDCK细胞用500μl从步骤3制备的病毒接种
  4. 将板在5%CO 2培养箱中在37℃下孵育45分钟
  5. 准备SeaPlaque DMEM(pDMEM)
    1. 将2×DMEM在37℃水浴中温热,并在搅拌下在约45-50℃的热板上将2%SeqPlaque琼脂糖熔化(*帽应稍微释放以防止压力累积)。
    2. 为了获得pDMEM,将预温热的2×DMEM和2%琼脂糖在无菌容器中以1:1的比例混合,并在室温下放置5-10分钟以冷却。
    3. 向pDMEM中加入TPCK-胰蛋白酶(终浓度:1μg/ml)
  6. 抽吸病毒接种物,并用1ml DPBS冲洗MDCK细胞一次。
  7. 向每个孔中加入1.5ml来自步骤7-c的pDMEM + TPCK-胰蛋白酶
  8. 将板在室温(RT)下放置10-15分钟,直到pDMEM固化
  9. 将板在37℃,5%CO 2下以反向取向(底部的盖子)温育3天。

 第5天(或直到斑块的可视化)

  1. 通过将1.5ml 10%福尔马林直接添加到琼脂糖顶部来固定MDCK细胞,并在RT下温育2小时。
  2. 吸出额外的福尔马林,并且使用实验室刮刀或等效工具,从孔中移除并且小心地丢弃琼脂糖而不干扰单层细胞。
    注意:在此步骤后,可以通过添加PBS覆盖细胞并在4°C下保存,以停止实验。
  3. MDCK细胞通过300μl0.5%Triton-X100在DPBS中在搅拌下在台上透化5分钟。
  4. 将细胞用1ml PBS漂洗两次
  5. 在室温下用摇动器摇动,用300μl5%的山羊血清在DPBS中封闭细胞30分钟
  6. 将细胞与H16-L10-4R5抗体(1:40)一起在300μl5%的山羊血清中在室温下摇动摇动培养1小时。
  7. 将细胞用1ml PBS漂洗两次
  8. 将细胞与碱性磷酸酶缀合的驴抗小鼠(1:1,000)在300μl5%山羊血清中在室温下在摇动器上摇动下孵育45分钟。
  9. 用1ml PBS冲洗细胞两次,用AP反应缓冲液冲洗细胞一次
  10. 准备BCIP/NBT溶液。
    1. 向每5ml的AP反应缓冲液中加入2滴溶液A,B和C
    2. 通过倒置管4-6次混合
  11. 向每个孔中加入300μlBCIP/NBT溶液。 将板放在摇摆器上在黑暗中30分钟或直到颜色的发展。
  12. 通过用ddH 2 O漂洗该板来终止反应。
  13. 将板在室温下风干。
  14. 计数斑块数,检查斑块形态并测量斑块的大小
    荧光,荧光聚焦测定(FFA)的替代可视化方法
    (从步骤18开始继续)
    • 19.1
    • 将细胞与山羊抗小鼠Alexa Fluor 488抗体一起温育 (1:1,000)在300μl的5%山羊血清中在室温下暗处培养45分钟。
    20.1用1ml PBS冲洗细胞两次。
    21.1细胞用500μlDPBS覆盖并在4℃下保存
    • 22.1
    • 检查斑块形态,并使用倒置荧光显微镜测量斑块的大小(图1) 注意:该协议已针对流感病毒进行了优化。修改 可能需要用于其他病毒。例如,要求的因素  对于多轮的病毒复制应该加入 pDMEM。


      图1. A /加利福尼亚/04/2009/H1N1重组病毒(rCA0409)的个体斑块的直径。 A.两个100个单个斑块的直径(μm)  不同的CA0409病毒,野生型(WT)和HA-S328Y突变体(HA328Y)。  通过Axiovert 200显微镜使用5x物镜和 通过IPlab3.6.5软件测量每个斑块的直径。统计  通过单尾学生t检验进行。 B.代表性形象 用Alexa Fluor 488染色的CA0409病毒噬菌斑,比例尺= 200 μm。图像由Sensicam使用IPlab 3.6.5(数据未公布)  (Tse等人,2013)。

食谱

  1. cDMEM(在0.2μm过滤单元中过滤)
    DMEM
    432.5毫升
    热灭活的胎牛血清         
    50 ml
    1 H HEPES                                               
    12.5 ml
    青霉素/链霉素
    5 ml
  2. 2x DMEM(在0.2μm过滤单元中过滤)
    DMEM粉末
    13.48克
    7.5%BSA馏分V
    26.7毫升
    1 M HEPES
    12.5 ml
    青霉素/链霉素
    5 ml
    ddH 2 2至500ml
  3. RPMI(以0.2μm过滤单位过滤)
    RPMI 1640粉末
    5.2克
    7.5%BSA馏分V / 13.3毫升
    1 M HEPES
    12.5 ml
    青霉素/链霉素
    5 ml
    ddH 2 2至500ml
  4. 0.5%Triton X-100
    Triton X- 100
    1 ml
    DPBS
    199 ml
  5. AP反应缓冲液
    Tris
    100 mM
    NaCl 
    100 mM
    MgCl 2
    5 mM
    用NaOH
    调节pH至9.5

致谢

我们感谢Jean K. Millet,Tamar Friling,Alice M. Hamilton和Whittaker实验室的所有成员进行有益的讨论。 我们还感谢Collins实验室在整个研究期间提供有用的建议。

参考文献

  1. Bussey,K.A.,Bousse,T.L.,Desmet,E.A.,Kim,B。和Takimoto,T。(2010)。 PB2残基271在哺乳动物宿主细胞中增强甲型流感病毒的聚合酶活性中起关键作用。/a> J Virol 84(9):4395-4406。
  2. Tse,L.V.,Marcano,V.C.,Huang,W.,Pocwierz,M.S.and Whittaker,G.R。(2013)。 纤溶酶介导的大流行H1N1流感病毒血凝素激活独立于病毒神经氨酸酶。 J Virol 87(9):5161-5169。   
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tse, L. V., Zhang, Y. and Whittaker, G. R. (2013). Immunoplaque Assay (Influenza Virus). Bio-protocol 3(21): e959. DOI: 10.21769/BioProtoc.959.
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