Isolation of Neutralizing Antibody
中和抗体的分离   

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PLOS Pathogens
Feb 2013

Abstract

Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. Then each of prepared HuMAbs is confirmed whether it can neutralize the specific virus by in vitro neutralization assay.

Keywords: Influenza (流感), Hemagglutinin (血凝素), Monoclonal antibody (单克隆抗体), Neutralization (中和), Vaccine (疫苗)

Materials and Reagents

  1. Human blood immunized by vaccination or natural infection against specific pathogen
  2. Human fusion partner SPYMEG cells (product of the Medical & Biological Laboratories Corporation, Ltd, Nagoya, Japan)
  3. Madin-Darby canine kidney (MDCK) cells provided from RIKEN cell bank
  4. MDCK-propagated Influenza B viruses (B/Florida/06/2004 and B/Malaysia/2506/2006) by the National Institute for Infectious Diseases, Japan
  5. Phosphate-buffered saline without Ca2+ and Mg2+ (PBS)
  6. HetaSep (STEMCELL Technologies, catalog number: 07906 )
  7. Ficoll-Paque PLUS (GE, catalog number: 17-1440-03 )
  8. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, catalog number: 11995 )
  9. Polyethylene glycol #1500 (PEG) (Roche Diagnostics, catalog number: 00-783-641-00 )
  10. Fecal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
  11. HAT supplement (Life Technologies, catalog number: 21060 )
  12. Antibody against the specific pathogen
  13. HT supplement (Life Technologies, catalog number: 11067 )
  14. Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
  15. Ethanol (Nacalai Tesque, catalog number: 14713-53 )
  16. Hybridoma SFM (Life Technologies, catalog number: 12045-084 )
  17. HiTrap Protein G HP (GE, catalog number: 17-0404-01 )
  18. BM condimed (Roche Diagnostics, catalog number: 10-663-573-001 )
  19. Heparin 5,000 units per 5 ml (Novo-Heparin, Mochida Phrrmaceutical)
  20. FITC-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, catalog number: 109-096-097 )
  21. DMEM-FBS medium (see Recipes)
  22. DMEM-HAT medium (see Recipes)
  23. DMEM-HT medium (see Recipes)
  24. PBS (see Recipes)

Equipment

  1. 75 cm2 cell culture flask (T-75 flask) (IWAKI PUMPS, catalog number: 3110-075 )
  2. 15- and 50-ml plastic tube (BD Biosciences,, catalog number: 352096 and 352070 )
  3. 6- and 10-cm culture dish (IWAKI PUMPS, catalog number: 3010-060 and 3020-100 )
  4. 6-, 12-, 24-, 48- and 96-well cell culture plate (IWAKI PUMPS, catalog number: 3810-006 , 3815-012 , 3820-024 , 3830-048 and 3860-096 )
  5. Light microscope
  6. Fluorescent microscope
  7. CO2 incubator (5% CO2, 37 °C)
  8. Incubator
  9. Peristaltic pump (Perista BioMini Pump; ATTO Corporation)
  10. Slide A Lyzer Dialysis Cassetes (MW = 10 K) (Thermo Fisher Scientific, model: 2160728 )
  11. Beaker
  12. Stirrer
  13. Stirrer bar
  14. Millex-HV Filter Unit 0.45 μm (EMD Millipore, model: SLHVJ13SL )
  15. 2.5-ml syringe
  16. Floater
  17. 21 G needle
  18. Centrifuge (TOMY DIGITAL BIOLOGY, model: LC-121 ; TS-7 rotor )

Procedure

Note: Every centrifugation step is performed at room temperature.

  1. Preparation of SPYMEG cells (SPYMEG cells, that are almost confluent in a T-75 flask, are needed per 10 ml blood sample)
    1. SPYMEG cells stored in freezed condition are thawed.
    2. Cells are transferred to a 15-ml tube and 10 ml DMEM-FBS are added.
    3. The cell suspension is centrifuged at 1,000 rpm for 5 min.
    4. The supernatants are aspirated and the cells in the precipitate are suspended with 20 ml DMEM-FBS.
    5. They are transferred to a T-75 flask.
    6. The cells are incubated in the CO2 incubator until the cells become confluent.
    7. Fresh 10 ml DMEM-FBS are added to the flask a day ahead of fusion.
    8. The cultured medium is aspirated and 10 ml DMEM is added in the flask.
    9. The cells are detached by tapping the flask strongly.
    10. The cells are transferred to a 50-ml tube.
    11. Tube is centrifuged at 1,000 rpm for 5 min.
    12. The supernatants are removed.
    13. The cell pellets are suspended with 10 ml DMEM.
    14. Tube is centrifuged at 1,000 rpm for 5 min.
    15. The supernatants are aspirated.
    16. The cell pellets are suspended with10 ml DMEM.

  2. Isolation of peripheral blood mononuclear cells (PBMCs)
    1. Fresh 10 ml blood is drawn from vaccinated donors or patients.
    2. Blood is collected in 15-ml tube including 1 drop of heparin.
    3. Blood is centrifuged at 1,000 rpm for 5 min.
    4. Plasma layer is removed.
    5. PBS is added up to 10 ml in the total volume.
    6. 3 ml undiluted HetaSep is added.
    7. The blood cells are mixed gently and stood for 1 h at room temperature.
    8. The upper layer is collected carefully and overlaid on 5 ml undiluted Ficoll in a 15-ml tube.
    9. Tube is centrifuged at 1,700 rpm for 1 h. (The brake is off).
    10. The upper layer is removed and the turbid intermediate layer is transferred to a new 50-ml tube.
    11. DMEM is added up to 20 ml in the total volume.
    12. The cells are mixed gently and centrifuged at 1,700 rpm for 5 min.
    13. The supernatants are removed.
    14. 20 ml DMEM is added.
    15. The cells are mixed gently and centrifuged at 1,700 rpm for 5 min.
    16. The supernatants are removed.
    17. 10 ml DMEM is added and the cells are mixed gently.

  3. Fusion
    1. 10 ml SPYMEG cells prepared in A16 and 10 ml PBMCs solution prepared in B17 are mixed gently in a 50-ml tube.
    2. The mixed cell suspension is centrifuged at 1,400 rpm for 5 min.
    3. The supernatants are removed.
    4. The 0.6 ml undiluted PEG is added gradually to the cell pellet and the cells are stirred gently (It should take 4 min).
    5. 10 ml DMEM is added gradually and the mixture is stirred gently (It should take 2 min).
    6. 10 ml DMEM-FBS is added (not need to mix).
    7. The tube is centrifuged at 1,400 rpm for 5 min.
    8. The supernatants are removed.
    9. 10 ml DMEM-FBS is added and the cells are mixed gently.
    10. Tube is centrifuged at 1,000 rpm for 5 min.
    11. The supernatants are removed.
    12. 10 ml DMEM-HAT is added and the cells are mixed gently.
    13. The cell solution is divided into 2 tubes.
    14. 45 ml DMEM-HAT is added to each of tube.
    15. 100 ml cell solution is seeded to 500 wells in 96-well plates (200 μl cell suspension per well).
    16. Plates are incubated in the CO2 incubator.
    17. 100 μl culture medium is changed to new DMEM-HAT every 3 to 4 days until fused cells grow up.

  4. Screening for the hybridoma producing antigen-specific antibody
    1. The infected MDCK cells are prepared for screening.
      1. MDCK cells are passaged in 2 x 104 cells per well (100 μl per well) in 96-well plates using MEM including 10% FBS a day ahead of infection.
      2. MDCK cells are washed with 100 μl PBS once. After aspiration of PBS, MDCK cells are adsorbed 50 μl influenza B virus propagated with MDCK cells at an MOI of 0.1 for 1 h in the CO2 incubator.
      3. 100 μl MEM is added to the wells.
      4. The plates are incubated in the CO2 incubator for 12 h.
      5. The medium is removed.
      6. The cells are washed with 100 μl PBS once and fixed with 200 μl absolute ethanol for 2 min at room temperature.
      7. The plates are air-dried and stored at -20°C until screening.
    2. The fixed MDCK cells prepared in D1 are adsorbed with the 75 μl cultured medium of fused cells prepared in C16 at 37 °C for 40 min.
    3. They are washed with 100 μl PBS three times with agitation for 5 min each.
    4. The plates are adsorbed with 75 μl FITC-conjugated anti-human IgG (1:500) and stood at 37 °C for 50 min.
    5. They are washed with 100 μl PBS three times with agitation for 5 min each.
    6. The fluorescent positive cells are observed under fluorescent microscope.
    7. The wells of hybridoma whose culture medium yield fluorescently positive cells are processed to cell cloning.

  5. Cloning of the hybridoma
    1. Each of wells including fluorescent positive cells in D7 is pipetted until the cells are detached.
    2. Cell number is counted using counting chamber.
    3. The cell suspension is adjusted to 1 cell per well in 96-well plate with DMEM-HT (200 μl per well).
    4. They are seeded to a 96-well plate and incubated in the CO2 incubator.
    5. 100 μl medium is changed to new DMEM-HT every 3 to 4 days until the cells grow up (Cell proliferation is checked using light microscope).
    6. Screening is performed again to select the antigen-specific hybridoma clone as described in D1 to D7.
    7. The hybridoma clones producing antigen-specific antibody are passaged to the larger-well plates (96-, 48-, 24-, 12- and 6-well plates and 6-cm culture dish) and ultimately grown up in 10-cm culture dish.

  6. Purification of human monoclonal antibody
    1. Ten 10-cm dishes including confluent hybridoma in DMEM-HT are prepared.
    2. The medium is changed to Hybridoma SFM.
    3. The cells are incubated in the CO2 incubator until the half of hybridoma cells die.
    4. The cultured medium are collected and centrifuged at 5,000 rpm for 30 min.
    5. The monoclonal antibody is purified by IgG-affinity column (HiTrap Protein G HP) according to the manufacturer’s instructions using Peristaltic pump.
    6. The purified antibody is filled in the syringe with needle.
    7. The antibody is injected to the dialysis cassette.
    8. The cassette with a floater is immersed in a beaker filled with 1,000 ml PBS and stirred at 4 °C for 2 h.
    9. 1,000 ml PBS is changed and the cassette is stirred again at 4 °C for 2 h.
    10. 1,000 ml PBS is changed and the cassette is stirred again at 4 °C overnight.
    11. The antibody in the cassette is collected and filtrated with a 0.45 μm filter.
    12. The concentration of the antibody is measured.

  7.  Viral neutralization assay
    1. MDCK cells are prepared in a 96-well plate a day ahead of viral neutralization assay as described in D1.
    2. The antibody at a concentration of 100 μg/ml is serially 4-fold diluted with MEM.
    3. 30 μl diluted antibodies or MEM as a control are incubated with 30 μl 200 focus-forming units of virus in the CO2 incubator for 1 h.
    4. MDCK cells are washed with 100 μl PBS and adsorbed with 30 μl mixture prepared in VII-3 in the CO2 incubator for 1 h.
    5. MDCK cells are washed with 100 μl PBS three times and added 100 μl MEM.
    6. They are incubated in the CO2 incubator for 12 h.
    7. The cells are washed with 100 μl PBS and fixed with 200 μl absolute ethanol for 2 min at room temperature.
    8. The cells are air-dried.
    9. The fixed MDCK cells are adsorbed with 75 μl 1st antibody (mouse serum infected with influenza B virus, 1:500) for detecting viral antigen and incubated at 37 °C for 30 min.
    10. They are washed with 100 μl PBS three times.
    11. They are adsorbed with 75 μl FITC-conjugated antibody for IgG of the 1st antibody and incubated at 37 °C for 45 min.
    12. They are washed with 100 μl PBS three times.
    13. The antigen positive cells are counted in each of wells under observation by fluorescent microscope (Figure 1).


      Figure 1. Fluorescent positive or negative cells observed by fluorescent microscope

    14. When the cell number is lower in the wells treated with antibody than in control (treated with PBS), the antibody is regarded as neutralizing monoclonal antibody. The lowest concentration of MAb that shows positive cell number to 50% compared with control is designated the VN50 titer. Low VN50 titer means that the MAb has strong neutralizing activity.

Recipes

  1. DMEM-FBS medium (500 ml)
    DMEM
    425 ml
    FBS
    75 ml
  2. DMEM-HAT medium (500 ml)
    DMEM
    401 ml
    FBS
    75 ml
    HAT supplement
    8 ml
    HT supplement
    1 ml
    BM condimed
    15 ml
  3. DMEM-HT medium (500 ml)
    DMEM
    405 ml
    FBS
    75 ml
    HT supplement
    5 ml
    BM condimed
    15 ml
  4. PBS (1,000 ml)
    NaCl
    8 g
    Na2HPO4.12H2O
    2.9 g
    KCl
    0.2 g
    KH2PO4
    0.2 g
    dH2O
    1,000 ml
    pH is not adjusted.
    Autoclaved

Acknowledgments

This work was supported in part by the Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS) (http://www.jst.go.jp/global/kadai/h2011_thailand.html); and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science to MY (#23790660).

References

  1. Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.

简介

使用单克隆抗体(MAbs)是用于表征特定病原体及其抗原性的已建立的实验室策略。 特别地,具有针对特定病毒的中和活性的人MAb(HuMAb)可具有潜在的治疗应用,并提供对于可能对于开发下一代针对该病毒的通用疫苗重要的人表位的重要信息。 除了用于鼠MAb制备的经典方法外,已经开发了几种制备HuMAbs的方法。 在这里,我们描述中和HuMAbs针对特定病毒的发展。 HuMAbs通过将接种的志愿者或患者的外周血单核细胞与融合伴侣细胞系(称为SPYMEG)融合而建立。 然后确认每个制备的HuMAb是否可以通过体外中和测定中和特异性病毒。

关键字:流感, 血凝素, 单克隆抗体, 中和, 疫苗

材料和试剂

  1. 通过疫苗接种或针对特定病原体的自然感染免疫的人血液
  2. 人融合伴侣SPYMEG细胞(Medical& Biological Laboratories Corporation,Ltd,Nagoya,Japan的产品)
  3. 由RIKEN细胞库提供的Madin-Darby犬肾(MDCK)细胞
  4. MDCK增殖的乙型流感病毒(B/Florida/06/2004和B/Malaysia/2506/2006),由日本国家传染病研究所提供
  5. 不含Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)
  6. HetaSep(STEMCELL Technologies,目录号:07906)
  7. Ficoll-Paque PLUS(GE,目录号:17-1440-03)
  8. Dulbecco改良Eagle培养基(DMEM)(Life Technologies,目录号:11995)
  9. 聚乙二醇#1500(PEG)(Roche Diagnostics,目录号:00-783-641-00)
  10. 粪牛血清(FBS)(MP Biomedicals,目录号:2917054)
  11. HAT supplement(Life Technologies,目录号:21060)
  12. 针对特定病原体的抗体
  13. HT补充剂(Life Technologies,目录号:11067)
  14. 最小必需培养基(MEM)(Sigma-Aldrich,目录号:M4655)
  15. 乙醇(Nacalai Tesque,目录号:14713-53)
  16. 杂交瘤SFM(Life Technologies,目录号:12045-084)
  17. HiTrap Protein G HP(GE,目录号:17-0404-01)
  18. BM condimed(Roche Diagnostics,目录号:10-663-573-001)
  19. 肝素5000单位/5ml(Novo-Heparin,Mochida Phrrhaceutical)
  20. FITC缀合的抗人IgG(Jackson ImmunoResearch Laboratories,目录号:109-096-097)
  21. DMEM-FBS培养基(参见配方)
  22. DMEM-HAT培养基(参见配方)
  23. DMEM-HT培养基(参见配方)
  24. PBS(请参阅配方)

设备

  1. 75cm 2细胞培养瓶(T-75烧瓶)(IWAKI PUMPS,目录号:3110-075)
  2. 15-和50-ml塑料管(BD Biosciences,目录号:352096和352070)
  3. 6-和10cm培养皿(IWAKI PUMPS,目录号:3010-060和3020-100)
  4. 6-,12-,24-,48-和96孔细胞培养板(IWAKI PUMPS,目录号:3810-006,3815-012,3820-024,3830-048和3860-096)
  5. 光学显微镜
  6. 荧光显微镜
  7. CO 2培养箱(5%CO 2,37℃)中培养。
  8. 孵化器
  9. 蠕动泵(Perista BioMini Pump; ATTO Corporation)
  10. Slide A Lyzer透析盒(MW = 10 K)(Thermo Fisher Scientific,型号:2160728)
  11. 烧杯
  12. 搅拌器
  13. 搅拌棒
  14. Millex-HV过滤器单元0.45μm(EMD Millipore,型号:SLHVJ13SL)
  15. 2.5毫升注射器
  16. 浮动
  17. 21 G针
  18. 离心机(TOMY DIGITAL BIOLOGY,型号:LC-121; TS-7转子)

程序

注意:每个离心步骤在室温下进行。

  1. 制备SPYMEG细胞(每10ml血液样品需要在T-75烧瓶中几乎汇合的SPYMEG细胞)
    1. 储存在冷冻条件下的SPYMEG细胞解冻
    2. 将细胞转移到15ml管中,加入10ml DMEM-FBS
    3. 将细胞悬浮液以1,000rpm离心5分钟。
    4. 吸出上清液,沉淀中的细胞用20ml DMEM-FBS悬浮
    5. 将它们转移到T-75烧瓶中
    6. 将细胞在CO 2培养箱中孵育直至细胞汇合
    7. 在融合前一天向烧瓶中加入新鲜的10ml DMEM-FBS
    8. 吸出培养基并在烧瓶中加入10ml DMEM
    9. 通过轻轻敲击烧瓶,可以分离细胞。
    10. 将细胞转移到50ml管中
    11. 管以1,000rpm离心5分钟。
    12. 除去上清液。
    13. 将细胞沉淀用10ml DMEM悬浮。
    14. 管以1,000rpm离心5分钟。
    15. 吸出上清液。
    16. 将细胞沉淀用10ml DMEM悬浮
  2. 外周血单核细胞(PBMC)的分离
    1. 从接种的供体或患者抽取新鲜的10ml血液
    2. 将血液收集在包含1滴肝素的15ml管中
    3. 血液以1,000rpm离心5分钟。
    4. 去除等离子体层。
    5. 加入PBS至总量为10ml。
    6. 加入3ml未稀释的HetaSep
    7. 将血细胞轻轻混合并在室温下放置1小时
    8. 小心地收集上层,并覆盖在15ml管中的5ml未稀释的Ficoll上
    9. 将管在1,700rpm离心1小时。 (制动器关闭)。
    10. 除去上层,将浑浊的中间层转移到新的50ml管中
    11. 在总体积中加入DMEM至多20ml
    12. 将细胞轻轻混合并以1,700rpm离心5分钟
    13. 除去上清液。
    14. 加入20ml DMEM
    15. 将细胞轻轻混合并以1,700rpm离心5分钟
    16. 除去上清液。
    17. 加入10ml DMEM,轻轻混合细胞
  3. 融合
    1. 将在A16中制备的10ml SPYMEG细胞和在B17中制备的10ml PBMC溶液在50ml管中轻轻混合。
    2. 混合细胞悬浮液在1,400rpm离心5分钟
    3. 除去上清液。
    4. 将0.6ml未稀释的PEG逐渐加入到细胞沉淀中,并将细胞轻轻搅拌(需要4分钟)。
    5. 逐渐加入10ml DMEM,并将混合物轻轻搅拌(需要2分钟)
    6. 加入10ml DMEM-FBS(不需要混合)。
    7. 将管在1400rpm离心5分钟。
    8. 除去上清液。
    9. 加入10ml DMEM-FBS,轻轻混合细胞
    10. 管以1,000rpm离心5分钟。
    11. 除去上清液。
    12. 加入10ml DMEM-HAT,轻轻混合细胞
    13. 细胞溶液分成2个管
    14. 向每个管中加入45ml DMEM-HAT
    15. 将100ml细胞溶液接种在96孔板中的500孔(每孔200μl细胞悬浮液)中
    16. 将板在CO 2培养箱中孵育
    17. 将100μl培养基每3至4天更换为新的DMEM-HAT,直到融合细胞生长
  4. 筛选产生抗原特异性抗体的杂交瘤
    1. 制备感染的MDCK细胞用于筛选。
      1. 在感染前一天,使用包括10%FBS的MEM在96孔板中以每孔2×10 4个细胞(每孔100μl)传代MDCK细胞。
      2. 将MDCK细胞用100μlPBS洗涤一次。 吸入PBS后,MDCK细胞吸附50μl用MDCK细胞以0.1的MOI繁殖的B型流感病毒在CO 2培养箱中1小时。
      3. 向孔中加入100μlMEM
      4. 将板在CO 2培养箱中温育12小时
      5. 介质被移除。
      6. 将细胞用100μlPBS洗涤一次,并在室温下用200μl无水乙醇固定2分钟
      7. 将板空气干燥并在-20℃下储存直至筛选
    2. 在D1中制备的固定MDCK细胞用在C16中制备的75μl融合细胞的培养基在37℃下吸附40分钟。
    3. 将它们用100μlPBS洗涤三次,每次搅拌5分钟
    4. 将平板用75μlFITC-结合的抗人IgG(1:500)吸附,并在37℃下静置50分钟。
    5. 将它们用100μlPBS洗涤三次,每次搅拌5分钟
    6. 在荧光显微镜下观察荧光阳性细胞
    7. 将培养基产生荧光阳性细胞的杂交瘤孔加工成细胞克隆
  5. 克隆杂交瘤
    1. 移取包括D7中的荧光阳性细胞的每个孔,直到细胞分离
    2. 使用计数室计数细胞数。
    3. 在96孔板中用DMEM-HT(每孔200μl)将细胞悬浮液调整至每孔1个细胞。
    4. 将它们接种到96孔板中并在CO 2培养箱中孵育
    5. 每3至4天将100μl培养基更换为新的DMEM-HT,直到细胞生长(使用光学显微镜检查细胞增殖)。
    6. 再次进行筛选以选择如D1至D7中所述的抗原特异性杂交瘤克隆
    7. 产生抗原特异性抗体的杂交瘤克隆传代至较大孔板(96孔,48孔,24孔,12孔板和6孔板和6cm培养皿),并最终在10cm培养皿 。

  6. 人单克隆抗体的纯化
    1. 制备10个10cm的包括DMEM-HT中的汇合杂交瘤的培养皿
    2. 培养基更换为Hybridoma SFM
    3. 将细胞在CO 2培养箱中培养直到杂交瘤细胞的一半死亡
    4. 收集培养基并以5,000rpm离心30分钟
    5. 根据制造商的说明使用蠕动泵通过IgG亲和柱(HiTrap Protein G HP)纯化单克隆抗体。
    6. 纯化的抗体用针头填充在注射器中
    7. 将抗体注射到透析盒中。
    8. 将具有浮子的盒浸入装有1,000ml PBS的烧杯中并在4℃下搅拌2小时。
    9. 改变1000ml PBS,并将该盒在4℃再次搅拌2小时。
    10. 改变1000ml PBS,并将该盒在4℃下再次搅拌过夜
    11. 收集盒中的抗体,并用0.45μm过滤器过滤
    12. 测量抗体的浓度。

  7.  病毒中和测定
    1. 在D1中所述的病毒中和测定前一天在96孔板中制备MDCK细胞
    2. 将浓度为100μg/ml的抗体用MEM连续稀释4倍
    3. 将30μl稀释的抗体或作为对照的MEM与30μl200焦点形成单位的病毒在CO 2培养箱中孵育1小时。
    4. 用100μlPBS洗涤MDCK细胞,并用在CO 2培养箱中VII-3中制备的30μl混合物吸附1小时。
    5. MDCK细胞用100μlPBS洗涤三次,并加入100μlMEM
    6. 将它们在CO 2培养箱中温育12小时
    7. 细胞用100μlPBS洗涤,并在室温下用200μl无水乙醇固定2分钟
    8. 细胞被空气干燥。
    9. 固定的MDCK细胞用75μl用于检测病毒抗原的第一抗体(小鼠血清感染乙型流感病毒,1:500)吸附,并在37℃下孵育30分钟。
    10. 将它们用100μlPBS洗涤三次。
    11. 将它们用75μlFITC-偶联的1号抗体的IgG抗体吸附,并在37℃孵育45分钟。
    12. 它们用100μlPBS洗涤三次。
    13. 在通过荧光显微镜观察的每个孔中对抗原阳性细胞计数(图1)

      图1.荧光显微镜观察到的荧光阳性或阴性细胞

    14. 当用抗体处理的孔中的细胞数低于对照(用PBS处理)中的细胞数时,抗体被认为是中和性单克隆抗体。 与对照相比,显示阳性细胞数至50%的MAb的最低浓度称为VN 50滴度。 低VN 50抗体滴度意指MAb具有强中和活性

食谱

  1. DMEM-FBS培养基(500ml)
    DMEM
    425 ml
    FBS
    75 ml
  2. DMEM-HAT培养基(500ml)
    DMEM
    401 ml
    FBS
    75 ml
    HAT补充
    8 ml
    HT补充
    1 ml
    BM约了
    15 ml
  3. DMEM-HT培养基(500ml)
    DMEM
    405 ml
    FBS
    75 ml
    HT补充
    5 ml
    BM约了
    15 ml
  4. PBS(1000ml)
    NaCl
    8克
    2 2
    2.9克
    KCl
    0.2 g
    KH 2 PO 4
    0.2 g
    dH 2 2 O 1000 ml
    pH值未调整。
    高压灭菌

致谢

这项工作得到日本科学技术机构/日本国际合作机构,可持续发展科学和技术研究伙伴关系(JST/JICA,SATREPS)的部分支持(http://www.jst.go.jp/global/kadai /h2011_thailand.html); 和日本科学促进会MY(#23790660)的青年科学家助学金(B)。

参考文献

  1. Yasugi,M.,Kubota-Koketsu,R.,Yamashita,A.,Kawashita,N.,Du,A.,Sasaki,T.,Nishimura,M.,Misaki,R.,Kuhara,M.,Boonsathorn,N 。,Fujiyama,K.,Okuno,Y.,Nakaya,T.and Ikuta,K.(2013)。 人类单克隆抗体广泛中和乙型流感病毒 PLoS Pathog < em> 9(2):e1003150。
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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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yasugi, M. and Ikuta, K. (2013). Isolation of Neutralizing Antibody. Bio-protocol 3(24): e1005. DOI: 10.21769/BioProtoc.1005.
  2. Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.
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