Human Blood Component Vaccinia Virus Neutralization Assay

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Molecular Therapy
Jun 2015



Many therapeutic viruses, such as oncolytic viruses, vaccines, or gene therapy vectors, may be administered by the intravenous route to maximize their delivery to target tissues. Blood components, such as antibody, complement and blood cells (such as neutrophils, monocytes, T cells, B cells or platelets) may result in viral neutralization and therefore reduce the therapeutic efficacy. This protocol will describe an in vitro assay by which to test the interaction of viruses with blood components. The effect of various factors can be isolated through fractionation. While whole blood can offer the most physiologically relevant snapshot, plasma can investigate the effects of antibody in concert with complement, and heat inactivated plasma will interrogate the effect of antibody alone.

Keywords: Complement (补充), Antibody (抗体), Neutralization (中和), Virus (病毒)

Materials and Reagents

  1. BD Vacutainer 7 ml Glass Serum Tubes, No Additive, Silicone-Coated Interior (BD, catalog number: 366431 )
  2. Eppendorf tubes (1.5 ml)
  3. T175 cm2 culture flasks (Corning Inc., catalog number: 431079 )
  4. 12-well plates culture plates (Corning Inc., catalog number: 3513 )
  5. 15 ml Falcon tubes (BD Biosciences, Falcon®, catalog number: 352096 )
    Note: Currently, it is “Thermo Fisher Scientific, FalconTM, catalog number: 352096 ”.
  6. Insulin syringes 27G1/2” (Terumo Medical Corporation, catalog number: SS05M2713 )

  7. 0.22 μm Millipore® StericupTM filter unit (Merck Millipore Corporation, catalog number: SCGPU05RE )
  8. Alcohol wipes
  9. U2OS human osteosarcoma tumor cells (ATCC), grown in complete DMEM (10% FBS)

  10. Vaccinia virus stock ≥ 5 x 106 pfu/ml
  11. 1x sterile phosphate-buffered saline (PBS) (Hyclone, catalog number: 21-031-CV )
    Note: Currently, it is “Mediatech, Inc., Corning, catalog number: 21-031-CV ”.
  12. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F1051 )
  13. Dulbecco’s modified Eagle medium (DMEM) culture media (Hyclone, catalog number: 10-031-CV )
    Note: Currently, it is “Mediatech, Inc., Corning, catalog number: 10-031-CV ”.
  14. 2x DMEM powder (Life Technologies, catalog number: 12800-017 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 12800-017 ”.
  15. Carboxymethylcellulose (CMC) (Sigma-Aldrich, catalog number: C5678-1KG )
  16. Sodium bicarbonate (Thermo Fisher Scientific, catalog number: S233-500 )
  17. 100x Penicillin-Streptomycin (GE Healthcare, Hyclone, catalog number: SV30010 )
  18. Refludan (Lepirudin)
  19. Crystal violet (Sigma-Aldrich, catalog number: C0775 )
  20. Methanol (Thermo Fisher Scientific, catalog number: A412P-4 )
  21. 2x DMEM solution (see Recipes)
  22. 3% Carboxymethylcellulose (CMC) (see Recipes)
  23. Titration overlay media (see Recipes)
  24. Crystal violet (see Recipes)


  1. Centrifuge (Thermo Fisher Scientific, model: ST40R ) 

  2. Biosafety cabinet 

  3. 37 °C, 5% CO2 incubator (Sanyo)
  4. ViCell (Beckman Coulter) or hemocytometer cell counter
  5. pH meter
  6. Heat blocks set to 37 °C and 56 °C
  7. BD Vacutainer® Push Button Blood Collection Set with Pre-Attached Holder (example 25G x 0.75 in) (BD, catalog number: 367336 )


  1. Preparation of cells for viral titration
    1. On the day prior to the neutralization experiment, 12 well plates should be set up with U2OS cells seeded at 2.5 x 105 cells/well. The U2OS cell line is a human osteosarcoma line that is highly infectable with Vaccinia virus and forms a very consistent monolayer, making it ideal for Vaccinia virus plaque assays. U2OS cells are cultured in DMEM with 10% FBS and should also be set up in the 12 well plates in the same media. The number of plates should correspond to the number of samples in which virus neutralization is to be assessed and account for duplicate or triplicate titrations.

  2. Collection of blood
    1. Clean the top of the vacutainer collection tube with an alcohol wipe.
    2. Venous blood should be collected by a trained phlebotomist into the vacutainer tube using a butterfly needle with connected vacutainer holder.
    3. Immediately after blood collection, using the insulin syringe, inject Refludan (70 μl of a 100x stock of Refludan to a final concentration of 50 μg/ml) through the rubber top of the tube. Invert several times (4-5 times) at room temperature to ensure proper mixing.

  3. Neutralization assay
    1. Prepare a 250 μl aliquot of whole blood per blood donor in an Eppendorf tube.
    2. Transfer remaining blood to a 15 ml falcon tube and spin at 800 x g for 5 min at room temperature to obtain plasma.
    3. Collect the plasma and prepare two 250 μl aliquots of plasma in Eppendorf tubes per blood donor.
    4. Heat inactivates one plasma sample (30 min at 56 °C) per blood donor to inactivate complement using either a heat block or a water bath.
    5. As a control to verify the amount of input virus under non-neutralizing conditions, prepare an additional tube with 250 μl of serum free media or PBS.
    6. Add 10 μl of prepared virus dilution (5 x 106 pfu/ml) to each sample and vortex very gently (final concentration is 2 x 105 pfu/ml). The total volume of each sample should be 260 μl.
    7. Incubate samples at 37 °C for one hour.

  4. Titration of infectious virus

    Figure 1. Titration procedure schematic

    1. Prepare 6 Eppendorf tubes with 900 μl of SFM per sample.
    2. Add 100 μl of sample to 900 μl of SFM. Make 1/10 serial dilutions with the remaining five tubes. (Dilution series will be 10-1 to 10-6.)
    3. Transfer 250 μl of diluted sample into the wells of the 12 well plate.
    4. Incubate for one hour at 37 °C. Every 15-20 min, plates should be rocked to ensure that no part of the well dries out during the minimal volume infection.
    5. Completely remove the inoculum by aspiration and add 1 ml of prepared titration overlay media per well.
    6. Two days after the virus neutralization experiment, visualize the plaque assay using a 0.1% crystal violet solution. Remove overlay by aspiration and wash wells with 1x PBS. Add 0.25 ml of 0.1% crystal violet solution to wells and incubate at room temperature for 5-10 min. Remove crystal violet and collect in a container for appropriate disposal. Wash the wells with tap water and dispose of this waste with crystal violet waste. Allow wells to air dry.
    7. Plaques are visible by the naked eye. Enumerate the number of plaques per well and multiply by the dilution factor.
    8. Data can be represented as Titer (pfu/ml) or as relative recovery to the control sample. Relative recovery can be calculated by dividing the titer of the test sample by the titer of the control non-neutralized sample (where virus was spiked into PBS or DMEM).

Representative data

Figure 2. Infectious Vaccinia virus recovery after incubation with blood, plasma or heat inactivated plasma from one representative human donor. Data can be represented as either titer (Log10 pfu/ml) as in panel A or as a proportion of virus recovered from the input (PBS or DMEM) as in panel B.


  1. Human blood must be collected with informed consent from all donors and according to a protocol approved by an institutional review board. Collection of information regarding previous exposures to the viruses in question, such as vaccination, may be important for interpreting experimental results and must be done according to an approved protocol. For example, in Evgin et al. (2015) blood donors for Vaccinia virus experiments were stratified according to self-reported vaccination status and date of birth relative to the end of the smallpox vaccination campaign.
  2. The protocol has been described for use with Vaccinia virus, however may be easily adapted using titration methods appropriate for the virus in question.
  3. The choice of anti-coagulant is important for assessment of the complement cascade. EDTA, Heparin and citrate will all interfere with complement activation. Hirudin and derivatives thereof, such as Refludan, are specific thrombin inhibitors and do not interfere with complement (Bexborn et al., 2009).
  4. Virus neutralization can be assessed at any concentration. Similarly, the volumes of blood or plasma can be adjusted as needed. This protocol uses the concentration of 2 x 105 pfu/ml which represents the clinical dose of 1 x 109 pfu in an estimated adult human blood volume of 5 L, as described in Evgin et al. (2015).
  5. This protocol can be adapted to use various inhibitors or blocking antibodies. For example, prior to step C6, peptide based complement inhibitors or inhibitory monoclonal antibodies were added in Evgin et al. (2015). An additional 15 min pre-incubation at 37 °C was included to ensure binding of the peptides or antibodies prior to the addition of virus.


  1. 2x DMEM (500 ml)
    Dissolve 1 package of DMEM powder in 500 ml of ddH2O
    Add 3.4 g of bicarbonate
    Adjust pH to 7.2
    Filter through a 0.22 μm Millipore® StericupTM filter unit or similar vacuum filtration system
    Add 100 ml FBS before making titration overlay media
    Stored at 4 °C
  2. 3% carboxymethylcellulose (CMC) (500 ml)
    Dissolve 15 g of CMC powder in 500 ml ddH2O
    Stored at room temperature
  3. Titration overlay media (500 ml)
    250 ml of 2x DMEM (+20% FBS)
    250 ml of 3% CMC
    5 ml 100x penicillin/streptomycin
    Stored at 4 °C
  4. Crystal violet
    Dissolve 1 g in 1 L of 80% methanol, 20% dH2O


The protocol was originally published in Evgin et al. (2015). This work was supported by the Terry Fox Research Foundation, the Ontario Institute for Cancer Research, the Ottawa Regional Cancer Foundation and the Canadian Institute for Health Research.


  1. Bexborn, F., Engberg, A. E., Sandholm, K., Mollnes, T. E., Hong, J. and Nilsson Ekdahl, K. (2009). Hirudin versus heparin for use in whole blood in vitro biocompatibility models. J Biomed Mater Res A 89(4): 951-959.
  2. Evgin, L., Acuna, S. A., Tanese de Souza, C., Marguerie, M., Lemay, C. G., Ilkow, C. S., Findlay, C. S., Falls, T., Parato, K. A., Hanwell, D., Goldstein, A., Lopez, R., Lafrance, S., Breitbach, C. J., Kirn, D., Atkins, H., Auer, R. C., Thurman, J. M., Stahl, G. L., Lambris, J. D., Bell, J. C. and McCart, J. A. (2015). Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques. Mol Ther 23(6): 1066-1076.


许多治疗性病毒,例如溶瘤病毒,疫苗或基因治疗载体,可以通过静脉内途径施用以最大化它们向靶组织的递送。 血液组分,例如抗体,补体和血细胞(例如嗜中性粒细胞,单核细胞,T细胞,B细胞或血小板)可导致病毒中和并因此降低治疗功效。 该方案将描述用于测试病毒与血液组分的相互作用的体外测定。 各种因素的影响可以通过分馏分离。 尽管全血可以提供最具生理学相关性的快照,但血浆可以研究抗体与补体一起的作用,而热灭活的血浆将会询问单独的抗体的作用。

关键字:补充, 抗体, 中和, 病毒


  1. BD Vacutainer 7 ml玻璃血清管,无添加剂,有机硅涂层内部(BD,目录号:366431)
  2. Eppendorf管(1.5ml)中
  3. T175cm 2培养瓶(Corning Inc.,目录号:431079)
  4. 12孔板培养板(Corning Inc.,目录号:3513)
  5. 15ml Falcon管(BD Biosciences,Falcon ,目录号:352096)
    注意:目前,"Thermo Fisher Scientific,Falcon TM ,目录号:352096" />
  6. 胰岛素注射器27G1/2"(Terumo Medical Corporation,目录号:SS05M2713)
  7. 0.22μmMillipore Stericup TM过滤器单元(Merck Millipore Corporation,目录号:SCGPU05RE)
  8. 酒精擦拭
  9. U2OS人骨肉瘤肿瘤细胞(ATCC),在完全DMEM(10%FBS)中生长
  10. 牛痘病毒储液≥5×10 6 pfu/ml
  11. 1x无菌磷酸盐缓冲盐水(PBS)(Hyclone,目录号:21-031-CV) 注意:目前,它是"Mediatech,Inc.,Corning,目录号:21-031-CV"。
  12. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F1051)
  13. Dulbecco改良的Eagle培养基(DMEM)培养基(Hyclone,目录号:10-031-CV)
  14. 2x DMEM粉末(Life technologies,目录号:12800-017)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:12800-017"
  15. 羧甲基纤维素(CMC)(Sigma-Aldrich,目录号:C5678-1KG)
  16. 碳酸氢钠(Thermo Fisher Scientific,目录号:S233-500)
  17. 100x青霉素 - 链霉素(GE Healthcare,Hyclone,目录号:SV30010)
  18. Refludan(Lepirudin)
  19. 结晶紫(Sigma-Aldrich,目录号:C0775)
  20. 甲醇(Thermo Fisher Scientific,目录号:A412P-4)
  21. 2x DMEM溶液(参见配方)
  22. 3%羧甲基纤维素(CMC)(参见配方)
  23. 滴定覆盖介质(参见配方)
  24. 水晶紫(见配方)


  1. 离心机(Thermo Fisher Scientific,型号:ST40R)
  2. 生物安全柜
  3. 37℃,5%CO 2培养箱(Sanyo)
  4. ViCell(Beckman Coulter)或血细胞计数器计数器
  5. pH计
  6. 加热块设置为37°C和56°C
  7. BD Vacutainer ?按钮血液收集套装带预连接座(示例25G x 0.75英寸)(BD,目录号:367336)


  1. 制备用于病毒滴定的细胞
    1. 在中和实验前一天,12孔板应该 ?用以2.5×10 5个细胞/孔接种的U2OS细胞建立。 U2OS细胞 ?线是高度可感染的人骨肉瘤系 牛痘病毒和形成非常一致的单层,使其理想 用于牛痘病毒噬菌斑测定。 U2OS细胞在DMEM中培养 10%FBS,并且也应在相同的12孔板中设置 媒体。板的数量应该对应于样品的数量 其中病毒中和将被评估和解释 重复或一式三份滴定。

  2. 血液收集
    1. 用酒精擦拭清洁真空采集管的顶部。
    2. 静脉血应由训练有素的抽血者收集 vacutainer管使用与被连接的vacutainer的蝴蝶针
    3. 收集血液后立即使用胰岛素 注射器,注射Refludan(70μl的100x库存的Refludan到最后 浓度为50μg/ml)通过管的橡胶顶部。倒置 在室温下数次(4-5次),以确保混合均匀

  3. 中和测定
    1. 在Eppendorf管中每个供血者准备250μl等份的全血
    2. 将剩余的血液转移到15ml falcon管中,并在室温下以800×g离心5分钟以获得血浆。
    3. 收集血浆,并准备两个250微升血浆等分试样在每个供血者的Eppendorf管。
    4. 热灭活每个血液供体的一个血浆样品(56℃下30分钟) 以使用加热块或水浴灭活补体。
    5. 作为控制来验证输入病毒的数量 非中和条件,准备一个额外的管与250微升 无血清培养基或PBS
    6. 加入10μl制备的病毒稀释液(5× ?10 6 pfu/ml)并非常轻柔地涡旋(终浓度 是2×10 5 pfu/ml)。每个样品的总体积应为260μl
    7. 在37℃孵育样品1小时。

  4. 感染性病毒滴定


    1. 准备6个Eppendorf管,每个样品有900μl的SFM
    2. 添加100 μl的样品加入到900μl的SFM中。使用1/10连续稀释 剩下的五个管。 (稀释系列将为10 -1 至10 -6 。)
    3. 将250μl稀释的样品转移到12孔板的孔中
    4. 在37℃孵育1小时。每15-20分钟,板应该 摇动以确保井的任何部分在最小化期间??干燥 体积感染。
    5. 通过抽吸完全去除接种物,并每孔加入1ml制备的滴定覆盖培养基
    6. 病毒中和实验两天后,可视化 使用0.1%结晶紫溶液进行噬斑测定。删除重叠式广告 抽吸和洗涤孔用1x PBS。加入0.25ml 0.1%的结晶 紫色溶液到孔中并在室温下孵育5-10分钟。 移除结晶紫并收集在适当的容器中 处置。用自来水冲洗井,并用这种废物处理 结晶紫浪费。允许井空气干燥。
    7. 斑块通过肉眼可见。枚举每孔的噬菌斑数,并乘以稀释因子。
    8. 数据可表示为滴度(pfu/ml)或相对回收率 ?对照样品。相对恢复可以通过除以 ?通过未中和的对照的滴度测试测试样品的滴度 样品(其中将病毒掺入PBS或DMEM中)。


图2.与来自一个代表性人类供体的血液,血浆或热灭活血浆孵育后的传染性牛痘病毒恢复。数据可表示为滴度(Log 10 10 pfu/ml) ),或如图B中从输入(PBS或DMEM)回收的病毒的比例。


  1. 人体血液必须在所有捐赠者的知情同意下并根据机构审查委员会批准的方案收集。关于先前暴露于所讨论的病毒(例如疫苗接种)的信息的收集对于解释实验结果可能是重要的,并且必须根据批准的方案进行。例如,在Evgin等人根据自我报告的疫苗接种状态和相对于天花疫苗接种运动结束的出生日期,对牛痘病毒实验的献血者进行分层。
  2. 已经描述了用于牛痘病毒的方案,然而可以使用适合于所讨论的病毒的滴定方法容易地适应。
  3. 抗凝剂的选择对于补体级联的评估是重要的。 EDTA,肝素和柠檬酸盐都会干扰补体激活。水蛭素及其衍生物例如Refludan是特异性凝血酶抑制剂,并且不干扰补体(Bexborn等人,2009)。
  4. 可以以任何浓度评估病毒中和。类似地,可以根据需要调节血液或血浆的体积。该方案使用2×10 5 pfu/ml的浓度,其代表在5L的估计成人人体血容量中的1×10 9 pfu的临床剂量,如描述在Evgin等人(2015)中
  5. 该方案可以适于使用各种抑制剂或阻断抗体。例如,在步骤C6之前,在Evgin等人(2015)中添加基于肽的补体抑制剂或抑制性单克隆抗体。在37℃下预孵育另外15分钟以确保肽或抗体在加入病毒之前结合。


  1. 2x DMEM(500ml)
    将1包DMEM粉末溶解在500ml ddH 2 O中 加入3.4克碳酸氢盐
    通过0.22μmMillipore Stericup TM 过滤器单元或类似的真空过滤系统过滤
    加入100 ml FBS,然后滴定覆盖介质
  2. 3%羧甲基纤维素(CMC)(500ml) 将15g CMC粉末溶解在500ml ddH 2 O中 高压灭菌器
  3. 滴定覆盖介质(500ml)
    250ml的2×DMEM(+ 20%FBS) 250ml 3%CMC
    5ml 100x青霉素/链霉素 储存在4°C
  4. 水晶紫
    将1g溶解在1L的80%甲醇,20%dH 2 O中。




  1. Bexborn,F.,Engberg,A.E.,Sandholm,K.,Mollnes,T.E.,Hong,J.and Nilsson Ekdahl,K。(2009)。 水蛭素与肝素用于全血在体外生物相容性模型。/a> J Biomed Mater Res A 89(4):951-959。
  2. Evgin,L.,Acuna,SA,Tanese de Souza,C.,Marguerie,M.,Lemay,CG,Ilkow,CS,Findlay,CS,Falls,T.,Parato,KA,Hanwell,D.,Goldstein,A Lopez,R.,Lafrance,S.,Breitbach,CJ,Kirn,D.,Atkins,H.,Auer,RC,Thurman,JM,Stahl,GL,Lambris,JD,Bell,JCand McCart,JA 2015)。 补体抑制防止免疫人类和食蟹猴中溶瘤性痘苗病毒中和。 Mol Ther 23(6):1066-1076。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Evgin, L. and Bell, J. (2015). Human Blood Component Vaccinia Virus Neutralization Assay. Bio-protocol 5(23): e1674. DOI: 10.21769/BioProtoc.1674.