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Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies
单克隆抗体介导的流感病毒微中和(MN)测定试验   

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本实验方案简略版
Nature Communications
Aug 2015

Abstract

The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol is to measure the ability of human monoclonal antibody to neutralize influenza virus by microneutralization assay.

Materials and Reagents

  1. Materials
    1. 96 well microtiter plates (Corning, catalog number: 3595 )
    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
      Note: Viruses were amplified in embryonated eggs or Madin–Darby canine kidney (MDCK) cells. Refer to Manual for the laboratory diagnosis and virological surveillance of influenza, WHO.
      1. H1N1
        A/Ohio/83
        A/Solomon Islands/2006
        A/Ohio/07/2009
        A/Texas/05/2009-RG15
        A/Texas/18/2009-RG18
        A/California/04/2009
      2. H2N2
        A/Ann Arbor/6/60 ca
      3. H5N1
        A/Vietnam/1203/04 (VNH5N1-PR8/CDC-RG)
        A/Anhui/01/2005(H5N1)-PR-IBCDC-RG6
      4. H9N2
        A/ck/HK/G9/97(H9N2)/PR8-IBCDC-2
        A/Green-winged teal/209/TX/2009
      5. H3N2
        A/Hong Kong/68
        A/Philippines/2/1982
        A/Beijing/353/89-X109-H3N2 PR8 reassortant
        A/Beijing/32/92-R-H3N2 PR8 reassortant
        A/Johannesburg/33/94 R-H3N2 PR8 reassortant
        A/Nanchang/933/95
        A/Sydney/5/97
        A/Panama/2007/99
        A/Wyoming/3/03.rg
        A/Brisbane/10/07
      6. H7N2
        A/turkey/Virginia/2002(H7N2)/ PR8-IBCDC-5
      7. H7N9
        A/Anhui/1/2013
        A/Shanghai/2/2013
    2. Cells
      Madin-Darby canine kidney (MDCK) cells (ATCC, catalog number: CCL-34 )
    3. Neutralization antibody
      Human monoclonal antibody, CT149 (Celltrion INC., South Korea) from convalescent patients infected with A(H1N1)pdm09
    4. Media
      1. DMEM (Invitrogen, catalog number: 11965-092 )
        Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 11965-092”.
      2. Fetal bovine serum (FBS) (VWR International, Hyclone, catalog number: SH30070.03 )
      3. Penicillin-streptomycin (Invitrogen, catalog number: 15140-122 )
        Note: Currently, it is (Thermo Fisher Scientific, Gibco™, catalog number: 15140-122)”.
      4. L-glutamine, 200 mM solution (Invitrogen, catalog number: 25030-081 )
        Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25030-081”.
      5. Bovine serum albumin (BSA) (fraction V, protease free) (Roche Diagnostics, catalog number: 0 3117332001 )
      6. HEPES, 1 M Buffer Solution (Invitrogen, catalog number: 15630-080 )
        Note: Currently, it is (Thermo Fisher Scientific, Gibco™, catalog number: 15630-080)”.
    5. Other reagents
      a. Antibody for ELISA
      1. Primary antibodies [Anti-nucleoprotein (NP) antibodies], Mouse Anti-Influenza A antibody (Merck Millipore Corporation, catalog number: MAB8257 ) and Anti-Influenza A antibody (Merck Millipore Corporation, catalog number: MAB8258 )
      2. Secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Kirkegaard & Perry Laboratories, Inc., catalog number: 074-1802 )
      b. Phosphate buffered saline (PBS) (Gibco, catalog number: 14190 )
      c. Tween-20 (Merck Millipore Corporation, catalog number: 8.17072 )
      d. Antibody diluent (Teknova, catalog number: D5120 )
      e. Acetone (Sigma-Aldrich, catalog number: 270725 )
      f. 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T0440 )
      g. Stop solution (Merck Millipore Corporation, catalog number: 109072 )
      Note: It is named “Sulfuric acid” on Merck Millipore Corporation website.
    6. Wash buffer (see Recipes)
    7. MDCK medium (see Recipes)
    8. Virus diluent media (see Recipes)

Equipment

  1. Haemacytometer (Hausser Scientific, catalog number: 1492 )
  2. Multichannel pipette (Gilson, model: PIPETMAN Neo® Multichannel )
  3. Incubator, 37 °C, 5% CO2 (Panasonic Corporation, Sanyo Electronics company, model: MCO-170AIC-PE )
  4. SpectraMax M5 multi-detection microplate reader system (Molecular Devices, model: M5 )

Procedure

  1. Prepare initial monoclonal antibody dilutions using virus diluent.
  2. Add 50 μl of virus diluent to all wells in column 1 to 10.
  3. Add 50 μl of the monoclonal antibody (1 mg/ml stock solution) to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette from the first well to each successive well from 1:1 to 1:128. Refer to Figure 1.
  4. Discard 50 μl after the last dilution.


    Figure 1. Serial dilution of test sample for MN assay. Add 50 μl of virus diluent to all wells in column 1 to 10 and add 50 μl of the sample to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette. Dilution factor of sample is 1 to 128.

  5. Cover and hold in 37 °C, 5% CO2 incubator while the diluted virus is being prepared. It is critical that the proper pH is maintained so that there will be no deleterious pH effects on the virus when it is added.
  6. Dilute the virus in virus diluent to the correct working dilution as 100 TCID50 /50 μl (TCID50: 50% Tissue culture infective dose, 100 TCID50: 100x TCID50).
  7. Add 50 μl of diluted virus to wells containing antibodies and the virus control (VC) wells (A12, B12, C12 and D12) and do not add virus to the cell control (CC) wells (E12, F12, G12 and H12) and do not add virus to the column of wells reserved for the virus back titration.
  8. Figure 2 showed detailed diagram for serial dilution of test antibody and back titration.


    Figure 2. Serial dilution of antibody and back titration. Serial dilution of test sample (green box) and virus back titration (yellow box) is 2-fold dilution by 50 μl + 50 μl. Virus control (blue box) is also 50 μl + 50 μl, but cell control (red box) is only 100 μl of diluent.

  9. Tap the plate gently to mix.
  10. Add 100 μl of virus diluent to the CC wells.
  11. Add 50 μl of virus diluent to the VC wells.
  12. In each assay, include a back titration of the working dilution of virus because neutralizing antibody titer is changed sensitively depend on virus titer.
  13. Add 50 μl of virus diluent to all wells in column 11.
  14. Add 50 μl of the working dilution of virus to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette from the first well to each successive well through well 12.
  15. Discard 50 μl from well 12.
  16. Add additional 50 μl of virus diluent to all wells for total volume to 100 μl.
  17. Cover and incubate at 37 °C, 5% CO2 for 1 h.
  18. After 1 h, add 100 μl of diluted MDCK cells (which should be 70-95% monolayer and low passage <30) to each well of microtiter plates. Each well contains 1.5 x 104 cells.
  19. Incubate at 37 °C, 5% CO2 for 18-20 h.
  20. After incubation, remove the medium from microtiter plates and wash the plates with 200 μl of PBS and then remove PBS (overturn the plate by hand to spill the PBS and then swap remaining liquid using paper towel).
  21. Add 100 μl of 80% cold acetone to each wells and incubate at room temperature (RT) for 10-12 min for fixation before ELISA.
  22. Remove the acetone (overturn the plate by hand to spill the acetone and then swap remaining liquid using paper towel), let the plates air-dry for 10 min or until dry.
  23. Dilute the primary antibody in antibody diluent to an optimum working dilution.
  24. Wash the plate 3x with 300 μl of the wash buffer and add 100 μl of diluted primary antibody (1:1,000).
  25. Cover and incubate for 1 h at RT.
  26. Dilute the secondary antibody in antibody diluent to an optimum working dilution
  27. Wash the plate 3x with 300 μl of the wash buffer and add 100 μl of diluted secondary antibody (1:2,000).
  28. Cover and incubate for 1 h at RT.
  29. Wash the plate 5x with 300 μl of the wash buffer and add 100 μl of freshly prepared TMB substrate.
  30. Incubate at RT until the color change in the VC wells is intense and the corresponding color change in the CC wells in minimal.
  31. Add 100 μl of stop solution.
  32. Read the absorbance (O.D.) of wells at 490 nm using microtiter plate spectrophotometer.
  33. The O.D. in the VC wells should be at least 0.8 and is typically in the range 1.0-1.5 though higher is acceptable.
  34. The O.D. in the CC wells must ≤ 0.2.
  35. The data are analyzed as follows.
    1. Evaluated to determine if the working dilution of virus used in the assay contained the desired amount of virus. The cut-off value for the virus back titration is the mean of the VC median and the CC median. This is the same cut-off value that is used to calculate neutralizing antibody titers. The dilution of the first well below the cut-off value is the back titration titer. The dilutions in the back titration wells are beginning at well A: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. In general, back titration titers of 16, 32, and 64 are acceptable.

      Example of back titration
      Median O.D. of CC = 0.2
      Median O.D. of VC= 1.0
      Mean of the VC median and the CC median = (0.2+1.0)/2= 0.6 = cut-off value
      O.D. of back titration wells (Table 1)

      Table 1. Example of O.D. of back titration wells
      Dilution
      1:1
      1:2
      1:4
      1:8
      1:16
      1:32
      1:64
      1:128
      O.D.
      2
      1.5
      1
      0.8
      0.5
      0.4
      0.3
      0.2

      1:16 (O.D.: 0.5) is the first well below the cut-off value (O.D. 0.6).
      Back titration titer is 1:16.

    2. Neutralizing antibody titers are determined by calculating the cut-off value to determine a 50% neutralizing antibody titer for each plate based on the equation: (median O.D. of VC + median O.D. of CC)/2 = X, where X = the 50% cut-off value. All values below or equal to X are positive for neutralization. Read each column which contained diluted antibody from the bottom, beginning at well H. Note the first well with an OD of less than the 50% cut-off. The reciprocal antibody dilution corresponding to that well is the 50% neutralization antibody titer for that antibody sample.

      Example of neutralizing antibody titer
      Median O.D. of CC = 0.2
      Median O.D. of VC = 1.0
      Mean of the VC median and the CC median = (0.2+1.0)/2 = 0.6 = cut-off value
      O.D. of back titration wells (Table 2)

      Table 2. Example of O.D. of neutralizing antibody wells
      Dilution
      1:1
      1:2
      1:4
      1:8
      1:16
      1:32
      1:64
      1:128
      O.D.
      0.1
      0.2
      0.3
      0.4
      0.5
      0.8
      1.5
      2.0

      1:4 (O.D.: 0.5) is the first well below the 50% cut-off value (O.D. 0.6).
      Neutralizing antibody titer is 1:16.

Recipes

  1. Wash buffer
    Phosphate buffered saline (PBS) with 0.3% Tween 20
  2. MDCK medium
    DMEM supplemented with
    10% fetal bovine serum (FBS)
    100 U/ml penicillin
    100 mg/ml streptomycin
    2 mM L-glutamine
    Sterilize by filtration
  3. Virus diluent media
    DMEM, supplemented with 1% bovine serum albumin (BSA) (fraction V, protease free) (prepared as a 10% w/v solution in dH2O, filter sterilized, and stored at 4-8 °C)
    100 U/ml penicillin
    100 mg/ml streptomycin
    20 mM HEPES
    Prepare fresh for each assay
    Sterilized by filtration

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. Klimov, A., Balish, A., Veguilla, V., Sun, H., Schiffer, J., Lu, X., Katz, J. M. and Hancock, K. (2012). Influenza virus titration, antigenic characterization, and serological methods for antibody detection. Methods Mol Biol 865: 25-51.
  2. Manual for the laboratory diagnosis and virological surveillance of influenza. WHO global influenza surveillance network.

简介

测试从单个人B细胞产生的人单克隆抗体,以表征其中和病毒感染性的能力。 微量中和测定法是用于检测流感病毒的病毒特异性中和抗体的高度灵敏和特异性的测定法。 该方案是测量人单克隆抗体通过微量中和测定中和流感病毒的能力。

材料和试剂

  1. 材料
    1. 96孔微量滴定板(Corning,目录号:3595)
    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
      注意:病毒在含胚卵中扩增   Madin-Darby犬肾(MDCK)细胞。 请参阅手册 实验室诊断和流感病毒学监测,WHO。
      1. H1N1
        A/Ohio/83
        A/Solomon Islands/2006
        A/Ohio/07/2009
        A/Texas/05/2009-RG15
        A/Texas/18/2009-RG18
        A/California/04/2009
      2. H2N2
        A/Ann Arbor/6/60 ca
      3. H5N1
        A/Vietnam/1203/04(VNH5N1-PR8/CDC-RG)
        A/Anhui/01/2005(H5N1)-PR-IBCDC-RG6
      4. H9N2
        A/ck/HK/G9/97(H9N2)/PR8-IBCDC-2
        A/Green-winged teal/209/TX/2009
      5. H3N2
        A/Hong Kong/68
        A/Philippines/2/1982
        A/Beijing/353/89-X109-H3N2 PR8重配体
        A/Beijing/32/92-R-H3N2 PR8重配体
        A/Johannesburg/33/94 R-H3N2 PR8重配体
        A /南昌/933/95
        A/Sydney/5/97
        A/Panama/2007/99
        A/Wyoming/3/03.rg
        A/Brisbane/10/07
      6. H7N2
        A/turkey/Virginia/2002(H7N2)/PR8-IBCDC-5
      7. H7N9
        A/Anhui/1/2013
        A/Shanghai/2/2013
    2. 单元格
      Madin-Darby犬肾(MDCK)细胞(ATCC,目录号:CCL-34)
    3. 中和抗体
      来自感染了A(H1N1)pdm09的恢复期患者的人单克隆抗体CT149(Celltrion INC。,South Korea)
    4. 媒体
      1. DMEM(Invitrogen,目录号:11965-092) 注意:目前,它是"Thermo Fisher Scientific,Gibco™,目录号:11965-092"。
      2. 胎牛血清(FBS)(VWR International,HycloneTM ,目录号:SH30070.03)
      3. 青霉素 - 链霉素(Invitrogen,目录号:15140-122)注意:目前,它是(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)"。
      4. L-谷氨酰胺,200mM溶液(Invitrogen,目录号:25030-081) 注意:目前,它是"Thermo Fisher Scientific,Gibco TM,目录号:25030-081"。
      5. 牛血清白蛋白(BSA)(级分V,无蛋白酶)(Roche Diagnostics,目录号:03117332001)
      6. HEPES,1M缓冲溶液(Invitrogen,目录号:15630-080) 注意:目前,它是(Thermo Fisher Scientific,Gibco TM,目录号:15630-080)"。
    5. 其他试剂
      一个。 ELISA抗体
      1. 一抗[抗核蛋白(NP)抗体],小鼠 抗流感A抗体(Merck Millipore Corporation,目录号: MAB8257)和抗流感A抗体(Merck Millipore Corporation, 目录号:MAB8258)
      2. 二抗,山羊抗小鼠IgG   缀合于辣根过氧化物酶(HRP)(Kirkegaard& Perry Laboratories,Inc.,目录号:074-1802)
      b。 磷酸盐缓冲盐水(PBS)(Gibco,目录号:14190)
      C。 Tween-20(Merck Millipore Corporation,目录号:8.17072) d。 抗体稀释剂(Teknova,目录号:D5120)
      e。 丙酮(Sigma-Aldrich,目录号:270725)
      F。 3,3',5,5'-四甲基联苯胺(TMB)(Sigma-Aldrich,目录号:T0440)
      G。 停止溶液(Merck Millipore Corporation,目录号:109072)
      注意:在默克密理博公司网站上命名为"硫酸"。
    6. 洗涤缓冲液(见配方)
    7. MDCK介质(参见配方)
    8. 病毒稀释培养基(见配方)

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. 培养箱,37℃,5%CO 2(Panasonic Corporation,Sanyo Electronics company,型号:MCO-170AIC-PE)
  4. SpectraMax M5多检测酶标仪系统(Molecular Devices,型号:M5)

程序

  1. 使用病毒稀释液制备初始单克隆抗体稀释液
  2. 向第1列到第10列的所有孔中加入50μl病毒稀释液。
  3. 加入50微升的单克隆抗体(1毫克/毫升储备溶液)到第一个井,并使用多通道移液器从第一个孔转移50微升2倍系列稀释液到每个连续的井从1:1到1 :128。参见图1.
  4. 最后稀释后弃去50μl。


    图1. MN测定的测试样品的系列稀释向第1至10列的所有孔中加入50μl病毒稀释液,并将50μl样品加入第一个孔中,制备2倍连续稀释通过转移50微升使用多通道移液器。样品的稀释因子为1至128.

  5. 覆盖并保持在37℃,5%CO 2培养箱中,同时制备稀释的病毒。至关重要的是保持适当的pH,以便在添加时对病毒没有有害的pH影响。
  6. 将病毒稀释液中的病毒稀释至正确的工作稀释度,为100 TCID 50 /50μl(TCID 50:50%组织培养感染剂量,100TCID 50:100×TCID 50 )。
  7. 加入50μl稀释的病毒到含有抗体和病毒对照(VC)孔(A12,B12,C12和D12)的孔,并且不添加病毒到细胞对照(CC)孔(E12,F12,G12和H12)不要为病毒回滴定保留的孔列添加病毒
  8. 图2显示了测试抗体的系列稀释和回滴定的详细图

    图2.抗体的系列稀释和反滴定。测试样品(绿色框)和病毒反滴定(黄色框)的系列稀释液用50μl+ 50μl稀释2倍。病毒对照(蓝色框)也是50微升+ 50微升,但细胞控制(红框)只有100微升的稀释剂。

  9. 轻轻轻拍以混合。
  10. 向CC孔中加入100μl病毒稀释液。
  11. 向VC孔中加入50μl病毒稀释液。
  12. 在每个测定中,包括对病毒的工作稀释的反滴定,因为中和抗体滴度敏感地改变取决于病毒滴度。
  13. 向第11列的所有孔中加入50μl病毒稀释液。
  14. 加入50μl病毒的工作稀释液到第一口井,并使用多通道移液器从第一口井转移50μl,通过井12每个连续的井,进行2倍系列稀释。
  15. 弃去孔12中的50μl。
  16. 向所有孔中再加入50μl病毒稀释液,总体积为100μl
  17. 盖上并在37℃,5%CO 2孵育1小时
  18. 1小时后,向微量滴定板的每个孔中加入100μl稀释的MDCK细胞(其应当是70-95%单层,低通量<30)。 每个孔含有1.5×10 4个细胞。
  19. 在37℃,5%CO 2孵育18-20小时
  20. 孵育后,从微量滴定板中取出培养基,用200μlPBS洗涤板,然后除去PBS(用手翻转板,溢出PBS,然后用纸巾交换剩余的液体)。
  21. 加入100微升80%冷丙酮到每个孔,孵育在室温(RT)10-12分钟,以便在ELISA之前固定。
  22. 取出丙酮(用手翻转板,溢出丙酮,然后用纸巾交换剩余的液体),让板空气干燥10分钟或直到干燥。
  23. 稀释抗体稀释液中的一抗以达到最佳工作稀释
  24. 用300μl洗涤缓冲液洗板3次,加入100μl稀释的一抗(1:1,000)
  25. 盖上并在室温下孵育1小时
  26. 稀释抗体稀释液中的第二抗体至最佳工作稀释度
  27. 用300μl洗涤缓冲液洗板3次,加入100μl稀释的二抗(1:2,000)
  28. 盖上并在室温下孵育1小时
  29. 用300μl洗涤缓冲液洗板5x,加入100μl新鲜制备的TMB底物。
  30. 在RT孵育,直到VC孔中的颜色变化剧烈,并且CC孔中的相应颜色变化最小。
  31. 加入100μl终止液
  32. 使用微量滴定板分光光度计在490nm处读取孔的吸光度(O.D.)
  33. 外径 在VC孔中应该至少为0.8,通常在1.0-1.5的范围内,尽管更高是可以接受的
  34. 外径 在CC井中必须≤0.2。
  35. 数据分析如下。
    1. 评估以确定是否使用的病毒的工作稀释 测定含有所需量的病毒。截止值 病毒回滴定是VC中值和CC中值的平均值。 这与用于计算中和的截止值相同 抗体滴度。第一孔的稀释度低于截止值 是反滴定滴度。在反滴定孔中的稀释液 以井A:1:1,1:2,1:4,1:8,1:16,1:32,1:64,1:128开始。在  一般,滴定滴定度为16,32和64是可以接受的
      回滴定示例
      中间O.D.的CC = 0.2
      中间O.D.的VC = 1.0
      VC中值和CC中值的平均值=(0.2 + 1.0)/2 = 0.6 =截止值
      O.D.的回滴定孔(表1)

      表1. O.D.的回滴定孔
      稀释
      1:1
      1:2
      1:4
      1:8
      1:16
      1:32
      1:64
      1:128
      O.D.
      2
      1.5
      1
      0.8
      0.5
      0.4
      0.3
      0.2

      1:16(O.D.:0.5)是低于临界值(O.D. 0.6)的第一口井 回滴定滴度为1:16。

    2. 通过计算中和抗体滴度来确定中和抗体滴度 截断值以确定每个的50%中和抗体滴度 基于等式:(VC的中值O.D. + CC的中值O.D.)/2 =   X,其中X = 50%截止值。 所有低于或等于X的值为 阳性的中和。 阅读每个含有稀释的柱子 抗体从底部开始,从孔H开始。注意第一个孔 小于50%截止值的OD。 互逆抗体稀释 对应于该孔是50%中和抗体滴度 该抗体样品
      中和抗体滴度的例子
      中间O.D. 的CC = 0.2
      中间O.D. 的VC = 1.0
      VC中值和CC中值的平均值=(0.2 + 1.0)/2 = 0.6 =截止值
      O.D. 的回滴定孔(表2)

      表2. O.D. 的中和抗体孔
      稀释
      1:1
      1:2
      1:4
      1:8
      1:16
      1:32
      1:64
      1:128
      O.D.
      0.1
      0.2
      0.3
      0.4
      0.5
      0.8
      1.5
      2.0

      1:4(O.D .: 0.5)是低于50%截止值(O.D. 0.6)的第一口井。
      中和抗体滴度为1:16。

食谱

  1. 洗涤缓冲液
    具有0.3%Tween20的磷酸盐缓冲盐水(PBS)
  2. MDCK介质
    DMEM补充有
    10%胎牛血清(FBS)
    100 U/ml青霉素
    100mg/ml链霉素 2mM l-谷氨酰胺 过滤灭菌
  3. 病毒稀释液
    DMEM,补充有1%牛血清白蛋白(BSA)(级分V,无蛋白酶)(制备为在dH 2 O中的10%w/v溶液,过滤灭菌, °C)
    100 U/ml青霉素
    100mg/ml链霉素 20 mM HEPES
    为每个测定准备新鲜的
    过滤灭菌

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,编号81401671),中国科学院战略重点研究计划(编号XDB08020100),中国国家大科技(授权号2015ZX09304005),GFG是NSFC创新研究组(授权号81321063)的首席主要研究员。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. Klimov,A.,Balish,A.,Veguilla,V.,Sun,H.,Schiffer,J.,Lu,X.,Katz,J.M.and Hancock,K。(2012)。 流感病毒滴定,抗原表征和抗体检测的血清学方法。 Methods Mol Biol 865:25-51。
  2. 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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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). Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies. Bio-protocol 6(11): e1829. DOI: 10.21769/BioProtoc.1829.
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