搜索

Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction
表面等离子共振分析抗原与抗体相互作用

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze the molecular interaction, enabling rapid and repetitive estimation with relatively small amount of sample. Here we describe a general protocol about SPR analysis on the interaction between viral antigen and human MAb (HuMAb) IgG. Anti-human Fcγ is first covalently crosslinked on the sensor chip by amine coupling, and then HuMAb of interest is immobilized via anti-Fcγ MAb IgG interaction as ligand. Antigen injected on the sensor chip causes the SPR change in time course as the result of association and dissociation. By analyzing the kinetics, association rate, dissociation rate, and dissociation constant are obtained.

Keywords: Molecular interaction(分子相互作用), Monoclonal antibody(单克隆抗体), IgG(IgG), Viral antigen(病毒抗原), Neutalization activity(neutalization活性)

Materials and Reagents

  1. Purified HuMAb (IgG)
  2. Purified antigen
  3. 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Nacalai tesque, catalog number: 17514-15 )
  4. Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 198-13765 )
  5. 2 mol/L sodium hydroxide solution (Nacalai Tesque, catalog number: 37441-45 )
  6. Sodium acetate, anhydrous (Wako Pure Chemical Industries, catalog number: 192-01075 )
  7. Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA.2Na) (Dojindo Molecular Technologies, catalog number: 345-01865 )
  8. Polyoxyethylene (20) sorbitan monolaurate (Wako Pure Chemical Industries, catalog number: 167-11515 )
  9. Goat Anti-Human Fcγ (Jackson ImmunoResearch Laboratories INC., catalog number: 109-005-008 )
  10. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific, catalog number: 22980 )
  11. N-hydroxysulfosuccinimide (NHS) (Thermo Fisher Scientific, catalog number: 24500 )
  12. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
  13. 2-ethanolamine (Nacalai tesque, catalog number: 23405-42 )
  14. Hydrochloric acid (Wako Pure Chemical Industries, catalog number: 080-01066 )
  15. Magnesium chloride hexahydrate (Wako Pure Chemical Industries, catalog number: 135-00165 )
  16. Sodium Chloride (Nacalai tesque, catalog number: 321319-45 )
  17. Glycine (Nacalai tesque, catalog number: 17109-35 )
  18. EDC/NHS solution
  19. 0.5 M HEPES buffer (pH 7.4) (see Recipes)
  20. 0.5 M EDTA (pH 8.0) (see Recipes)
  21. 20% (v/v) polysorbate 20 (see Recipes)
  22. 10x HBS-E (see Recipes)
  23. 1x HBS-EP (see Recipes)
  24. 400 mM EDC (see Recipes)
  25. 100 mM NHS (see Recipes)
  26. 1 M 2-ethanolamine (pH 8.5) (see Recipes)
  27. 50 mM NaOH (see Recipes)
  28. 10 mM Acetate buffer (pH 4.0, 4.5, 5.0, 5.5) (see Recipes)
  29. Glycine buffer 10 mM (pH 1.8, 2.0 2.2, 2.4) (see Recipes)
  30. 1 M NaCl (see Recipes)
  31. 3 M MgCl2 (see Recipes)

Equipment

  1. Instrument for SPR analysis, e.g. Biacore T200 (GE) or ProteOn XPR36 (Bio-Rad Laboratories)
  2. Sensor chip (with carboxyl groups available for the amine coupling reaction), e.g. Series S Sensor Chip CM5 (GE, catalog number: BR-1005-30 ) for Biacore T200 or ProteOn GLM Sensor Chip (Bio-Rad Laboratories, catalog number: 176-5012 ) for ProteOn XPR36
  3. Vacuum pump
  4. 0.22 μm PES PLUS bottom top filter (IWAKI PUMPS, catalog number: 8024-045 )
  5. 0.22 μm Millex-GV syringe filter unit (Millipore, catalog number: SLGV033RS )
  6. 20 ml syringe (Thermo Fisher Scientific, catalog number: SS-20ESZ )
  7. Centrifuge
  8. 1.5 ml centrifuge tube (Ina-optika corporation, catalog number: CF-0150 )
  9. Spectra/Por 3 Dialysis trial kit (SPECTRUM® LABORATORIES, catalog number: 132720T )
  10. Stir bar and stirrer
  11. UV spectrometer
  12. pH meter

Procedure

  1. Preparation of reagents and sample
    1. Prepare reagents (HBS-EP buffer, 400 mM EDC, 100 mM NHS, 50 mM NaOH, 1 M 2-ethanolamine-HCl, glycine buffers and acetate buffers).
      Wash dialysis membrane by 300 ml of dH2O and dialyze antigen in 1 L of HBS-EP buffer on stirrer at 4 °C for at least 8 h. The HBS-EP buffer should be changed to fresh one twice with at least 8 h interval.
      Notes:
      1.  Imbalance in the refractive index between antigen solution and running buffer will cause high background signal (called bulk effect) and perturb the SPR analysis.
      2.  The concentration of antigen is recommended to be 5 μM or higher, because the dissociation constant for weak antigen-antibody interaction sometimes reaches to 10-6 M range and in that case SPR experiment requires relatively high concentration of antigen, that is 5 μM or more.
    2. Determine antigen concentration by ultraviolet UV spectroscopy or standard BCA analysis.

  2. Immobilization of anti-human Fcγ on sensor chip
    1. Set sensor chip into the SPR instrument.
      Note: In all steps dealing with the instrument, you should follow the instrument’s manual.
    2. Set the 1 L of HBS-EP buffer as running buffer.
    3. Prepare 300 μl l of ligand solutions: 100 μg/ml anti-human Fcγ solutions of four different pH values (4.0, 4.5, 5.0 and 5.5) by diluting with the 10 mM acetate buffer.
    4. Inject ligand solutions and determine the optimum pH condition in which the highest response level is observed (Figure 1a).
      Note: When there is little or no difference, select higher pH in order to avoid ligand deterioration and to promote coupling reaction. Practically, pH 4.0 or 4.5 is selected for immobilization of anti-human Fcγ, though you should determine for your SPR system.
    5. Initialize the chip surface by injecting 30 μl of 50 mM NaOH.
    6. Activate the chip surface by injecting 70 μl of 1:1 EDC/NHS mix.
    7. Inject anti-human Fcγ solution and check the baseline increment. Repeat injection until the increment becomes undetectable.
      Note: In this step anti-human Fcγ should immobilize as much as possible. Usually, approx. 8,000 (Resonance Unit, RU) increment is obtained.
    8. Inject 70 μl of 1 M 2-ethanolamine-HCl solution to block the active carboxyl groups remained on the sensor chip.
      Note: If you want stop the experiment in this step, eject the sensor chip from the instrument and store at 4 °C in HBS-EP buffer to prevent drying out.

  3. Check the antigen-MAb interaction and design experiment.
    1. Prepare 1 ml of 1 μg/ml MAb solution by diluting with HBS-EP buffer.
      Note: The original MAb solution should not include any reagents which perturb the interaction between MAb and anti-human Fcγ, for example, 100 mM glycine buffer of pH 2.4 or lower.
    2. Inject target MAb and check the immobilization amount as the increment of baseline level.
      Note: For SPR analysis, smaller immobilization amount is preferred as far as the response against antigen gives sufficient signal.
    3. Inject 30 μl of 10 mM glycine solutions of different pH values 1.8, 2.0, 2.2, and 2.4. By checking the decrement of the baseline level, determine the MAb-stripping condition (pH, injection period, number of injections, period required for baseline stabilization).
      Note: The baseline level should be close to the level before MAb immobilization. An example of practical condition is as follows: Inject 30 μl of 10 mM Glycine pH 2.0 thrice and wash by running buffer for 600 seconds as baseline stabilization period.
    4. Prepare 1 ml of 1 μM antigen solution by diluting with HBS-EP buffer.
    5. Clarify the antigen solution by centrifugation at 15,000 x g for 5 min at room temperature.
    6. Inject 60 μl of the antigen solution and check the SPR response.
      Note: The minimum response level is 1 RU or less as far as the response curve is recognizable, though it apparently depends on the baseline stability, in other words, stability of the SPR system.
    7. Determine antigen-stripping condition by injecting 60 μl of 1 M NaCl or 3 M MgCl2.
      Note: As other option, you can remove the whole antigen-MAb with glycine buffer and retry from the MAb immobilization step. Antigen-stripping condition is not necessarily required depending on the experimental design.
    8. Prepare 1 ml of antigen solutions of at least 5 different concentrations by 1:1 serial dilution with HBS-EP buffer.
    9. Determine the experimental design considering the following points.
      1. MAb immobilization condition.
      2. Injection period for the antigen solution and washing period.
        Notes:
        1. They are corresponding to the lengths of association and dissociation phases, respectively. Set appropriate values to obtain significant signal level and sufficient curvature.
        2. In some cases antigen dissociates very slowly from MAb, and then we need to set very long washing period. When no dissociation is observed, try reducing the immobilizing level of MAb.
      3. Antigen-stripping condition.
      4. Design of blank experiment: without MAb immobilization and/or injecting HBS-EP buffer in replace of antigen solution.
      5. Setting for individual flow paths.
        Note: Usually, SPR system has multiple flow paths useful for blank experiment and for examination of multiple MAbs at once.

  4. Curve analysis
    1. Collected experimental data is analyzed according to the fitting algorism supplied by vendor of the SPR system (For example, see Figure 1c). Usually, 1:1 Langmuir fitting model is employed.
      Note: Check the chi-square value and quality index supplied by the software.


      Figure 1. Practical example of the SPR experiment.
      1. Determination of the pH value for amine-coupling immobilization of anti-human Fcγ. pH 4.0 showed the highest response, however, it also showed signs of leveling off. Then, we have chosen pH 4.5 in this case. Open arrowhead and closed arrowhead indicate the start and end points of the injection of anti-human Fcγ solution, respectively.  
      2. Immobilization of MAb on the sensor chip via the interaction between anti-human Fcγ and human MAb. Open arrowhead and closed arrowhead indicate the start and end points of the injection of human MAb solution, respectively. The increment of baseline level (in this case, approx. 10 RU) represents the immobilized amount of MAb.
      3. Representative SPR response for the interaction between influenza B hemagglutinin (HA) and a human anti-HA antibody. Open arrowheads indicate the injection of HA solution and correspond to the association phase. Closed arrowhead indicates the end of the final injection and corresponds to the start of the dissociation phase. Fitting curve obtained by the 1:1 Langmuir fitting model was also shown by gray line.

Notes

  1. Usually SPR analysis is performed under an integrated system in which equipment (sensor chip, buffer bottle, sample tube, etc), experimental design, analysis algorism and even in reagents are all supplied for using that instrument. We just described here a general procedure of SPR experiment for antigen-MAb interaction analysis. Please follow the manual of your instrument and/or contact the vendor for more detailed protocol.

Recipes

  1. 0.5 M HEPES buffer (pH 7.4) (500 ml)
    Mix 59.6 g of HEPES powder with 400 ml dH2O
    Adjust pH to 7.4 with NaOH
    Add dH2O to 500 ml
    Filtrate by 0.22 μm bottom top filter and vacuum pump
  2. 0.5 M EDTA (pH 8.0) (500 ml)
    Mix 93.1 g of EDTA.2Na with 400 ml dH2O
    Adjust pH to 8.0 with NaOH
    Add dH2O to 500 ml
    Filtrate by 0.22 μm bottom top filter and store at room temperature
  3. 20 % (v/v) polysorbate 20 (500 ml)
    Add 400 ml dH2O to 100 ml polyoxyethylene (20) sorbitan monolaurate
  4. 10x HBS-E (500 ml)
    Mix 100 ml 0.5 M HEPES buffer, pH 7.4
    30 ml 0.5 M EDTA, pH 8.0
    44 g NaCl
    Add dH2O to 500 ml
  5. 1x HBS-EP (4 L)
    10x HBS-E buffer 400 ml
    Add dH2O to 4 L
    Filtrate by 0.22 μm bottom top filter and vacuum pump
    Add 1 ml 20% (v/v) polysorbate 20
  6. 400 mM EDC (10 ml)
    Dissolve 750 mg EDC in 10 ml dH2O
    Dispense 200 μl into 1.5 ml tubes and store at -20 °C
  7. 100 mM NHS (10 ml)
    Dissolve 115 mg NHS in 10 ml dH2O
    Dispense 200 μl into 1.5 ml tubes and store at -20 °C
  8. 1 M 2-ethanolamine (pH 8.5) (50 ml)
    Dilute 3.0 ml of 2-ethanolamine with dH2O to 40 ml
    Adjust pH to 8.5 by HCl
    Add dH2O to 50 ml
    Dispense 1 ml into 1.5 ml tubes and store at 4 °C
  9. 50 mM NaOH (10 ml)
    Dilute 250 μl of 2 M NaOH solution with dH2O to 10 ml.
    Dispense 1 ml into 1.5 ml tubes and stored at room temperature
  10. 10 mM Acetate buffer (pH 4.0, 4.5, 5.0, 5.5) (50 ml each)
    Mix 8.2 g sodium acetate with 100 ml dH2O to prepare 1 M acetate-Na
    Dilute 5.7 ml of acetate with 100 ml dH2O to prepare 1 M acetate
    Prepare 10 mM acetate-Na and 10 mM acetate by 100-fold dilution with dH2O
    Adjust pH by mixing 10 mM acetate and 10 mM acetate-Na
    Filtrate by 0.22 μm filter and syringe
  11. Glycine buffer 10 mM (pH 1.8, 2.0 2.2, 2.4) (50 ml each)
    Mix 0.30 g glycine with dH2O and add dH2O to 200 ml
    Dispense 50 ml glycine solution into 100 ml beaker
    Adjust pH by adding HCl
    Add dH2O to 100 ml
    Filtrate by 0.22 μm filter and syringe
  12. 1 M NaCl
    Mix 2.9 g NaCl with dH2O to 50 ml
    Filtrate by 0.22 μm filter and syringe
  13. 3 M MgCl2
    Mix 30.5 g MgCl2.6H2O with dH2O and adjust volume to 50 ml
    Filtrate by 0.22 μm filter and syringe

Acknowledgments

This protocol was established by reference to the product manuals of Biacore T200 (GE-Healthcare) and ProteOn XPR36 (BIO-RAD).

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

单克隆抗体(MAb)与其识别的抗原之间的分子相互作用是导致中和活性的基本事件。 它们的结合亲和力的估计给出了表征MAb和产生更有效的MAb的有益信息。 表面等离子体共振(SPR)分析是分析分子相互作用的有力工具,使得能够用相对少量的样品进行快速和重复的估计。 在这里我们描述关于病毒抗原和人类MAb(HuMAb)IgG之间的相互作用的SPR分析的一般协议。 抗人Fcγ首先通过胺偶联在传感器芯片上共价交联,然后通过抗FcγMAbIgG相互作用作为配体固定目标HuMAb。 注射在传感器芯片上的抗原导致作为结合和解离的结果的时间过程中的SPR变化。 通过分析动力学,获得结合速率,解离速率和解离常数。

关键字:分子相互作用, 单克隆抗体, IgG, 病毒抗原, neutalization活性

材料和试剂

  1. 纯化的HuMAb(IgG)
  2. 纯化抗原
  3. 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)(Nacalai tesque,目录号:17514-15)
  4. 氢氧化钠(Wako Pure Chemical Industries,目录号:198-13765)
  5. 2mol/L氢氧化钠溶液(Nacalai Tesque,目录号:37441-45)
  6. 乙酸钠,无水(Wako Pure Chemical Industries,目录号:192-01075)
  7. 乙二胺-N,N,N',N'-四乙酸二钠盐二水合物(EDTA 2Na)(Dojindo Molecular Technologies,目录号:345-01865)
  8. 聚氧乙烯(20)山梨糖醇酐单月桂酸酯(Wako Pure Chemical Industries,目录号:167-11515)
  9. 山羊抗人Fcγ(Jackson ImmunoResearch Laboratories INC。,目录号:109-005-008)
  10. 1-乙基-3- [3-二甲基氨基丙基]碳二亚胺盐酸盐(EDC)(Thermo Fisher Scientific,目录号:22980)
  11. (NHS)(Thermo Fisher Scientific,目录号:24500)。
  12. 乙酸(Wako Pure Chemical Industries,目录号:017-00256)
  13. 2-乙醇胺(Nacalai tesque,目录号:23405-42)
  14. 盐酸(Wako Pure Chemical Industries,目录号:080-01066)
  15. 氯化镁六水合物(Wako Pure Chemical Industries,目录号:135-00165)
  16. 氯化钠(Nacalai tesque,目录号:321319-45)
  17. 甘氨酸(Nacalai tesque,目录号:17109-35)
  18. EDC/NHS溶液
  19. 0.5 M HEPES缓冲液(pH 7.4)(见配方)
  20. 0.5 M EDTA(pH 8.0)(参见配方)
  21. 20%(v/v)聚山梨酯20(参见配方)
  22. 10x HBS-E(见配方)
  23. 1x HBS-EP(参见配方)
  24. 400 mM EDC(见配方)
  25. 100 mM NHS(参见配方)
  26. 1 M 2-乙醇胺(pH 8.5)(参见配方)
  27. 50 mM NaOH(见配方)
  28. 10mM乙酸盐缓冲液(pH 4.0,4.5,5.0,5.5)(参见Recipes)
  29. 甘氨酸缓冲液10 mM(pH 1.8,2.0 2.2,2.4)(参见配方)
  30. 1 M NaCl(见配方)
  31. 3 M MgCl <2> (参见配方)

设备

  1. 用于SPR分析的仪器,例如Biacore T200(GE)或ProteOn XPR36(Bio-Rad Laboratories)
  2. 传感器芯片(具有可用于胺偶联反应的羧基),例如用于Biacore T200的系列S传感器芯片CM5(GE,目录号:BR-1005-30)或ProteOn GLM传感器芯片(Bio- Rad Laboratories,目录号:176-5012)用于ProteOn XPR36
  3. 真空泵
  4. 0.22μmPES PLUS底部过滤器(IWAKI PUMPS,目录号:8024-045)
  5. 0.22μmMillex-GV注射器过滤单元(Millipore,目录号:SLGV033RS)
  6. 20ml注射器(Thermo Fisher Scientific,目录号:SS-20ESZ)
  7. 离心机
  8. 1.5ml离心管(Ina-optika corporation,目录号:CF-0150)
  9. Spectra/Por 3透析试剂盒(SPECTRUM LABORATORIES,目录号:132720T)
  10. 搅拌棒和搅拌器
  11. 紫外光谱仪
  12. pH计

程序

  1. 试剂和样品的制备
    1. 制备试剂(HBS-EP缓冲液,400mM EDC,100mM NHS,50mM NaOH,1M 2-乙醇胺-HCl,甘氨酸缓冲液和乙酸盐缓冲液)。 在4℃下在搅拌器上在1L HBS-EP缓冲液中用300ml dH 2 O和透析抗原洗涤透析膜至少8小时。 HBS-EP缓冲液应更换为新鲜的一次,间隔至少8小时 注意:
      1. 抗原溶液和运行缓冲液之间的折射率不平衡将导致高背景信号(称为体效应)并干扰SPR分析。
      2. 抗原的浓度推荐为5μM或更高,因为弱抗原 - 抗体相互作用的解离常数有时达到10-6M范围,并且在这种情况下SPR实验需要相对高浓度的抗原,即 为5μM以上。
    2. 通过紫外线UV光谱或标准BCA分析确定抗原浓度
  2. 抗人Fcγ在传感器芯片上的固定化
    1. 将传感器芯片装入SPR仪器。
      注意:在处理仪器的所有步骤中,应遵循仪器的手册。
    2. 将1 L HBS-EP缓冲液设置为运行缓冲液。
    3. 通过用10mM乙酸盐缓冲液稀释制备300μl配体溶液:100μg/ml四种不同pH值(4.0,4.5,5.0和5.5)的抗人Fcγ溶液。
    4. 注射配体溶液并确定观察到最高反应水平的最佳pH条件(图1a) 注意:当几乎没有或没有差异时,选择较高的pH以避免配体变质并促进偶联反应。 实际上,选择pH 4.0或4.5用于抗人Fcγ的固定,尽管您应该为您的SPR系统确定。
    5. 通过注入30μl的50mM NaOH初始化芯片表面
    6. 通过注射70μl1:1 EDC/NHS混合物激活芯片表面
    7. 注射抗人Fcγ溶液并检查基线增量。重复注射,直到增量变得无法检测。
      注意:在该步骤中,抗人Fcγ应尽可能固定。通常,约。 8,000(共振单位,RU)增量。
    8. 注入70μl的1M 2-乙醇胺-HCl溶液以阻断传感器芯片上剩余的活性羧基 注意:如果您想在此步骤中停止实验,请将传感器芯片从仪器中取出,并在4°C下在HBS-EP缓冲液中储存,以防止干燥。

  3. 检查抗原-MAb相互作用和设计实验。
    1. 通过用HBS-EP缓冲液稀释制备1ml的1μg/ml MAb溶液 注意:原始MAb溶液不应包括任何干扰MAb和抗人Fcγ之间相互作用的试剂,例如pH 2.4或更低的100mM甘氨酸缓冲液。
    2. 注射目标MAb,并检查固定量作为基线水平的增量 注意:对于SPR分析,较小的固定量是优选的,只要对抗原的反应给出足够的信号。
    3. 注入30μl不同pH值1.8,2.0,2.2和2.4的10mM甘氨酸溶液。通过检查基线水平的减量,确定MAb剥离条件(pH,注射时间,注射次数,基线稳定所需的时间)。
      注意:基线水平应接近MAb固定前的水平。实施条件的实例如下:注入30μl10mM甘氨酸pH2.0三次,并通过运行缓冲液洗涤600秒作为基线稳定期。
    4. 通过用HBS-EP缓冲液稀释制备1ml1μM抗原溶液
    5. 通过在室温下以15,000×g离心5分钟澄清抗原溶液。
    6. 注射60μl抗原溶液并检查SPR反应。
      注意:尽管响应曲线是可识别的,但最小响应级别为1 RU或更小,但它显然取决于基线稳定性,换句话说,SPR系统的稳定性。
    7. 通过注射60μl的1M NaCl或3M MgCl 2来确定抗原剥离条件。
      注意:作为其他选择,您可以用甘氨酸缓冲液去除整个抗原-MAb,并从MAb固定化步骤重试。根据实验设计,抗原剥离条件不是必需的。
    8. 准备1毫升至少5种不同浓度的抗原溶液,用HBS-EP缓冲液按1:1连续稀释
    9. 考虑以下几点确定实验设计。
      1. MAb固定条件。
      2. 抗原溶液的注射期和洗涤期 注意:
        1. 它们分别对应于缔合和解离期的长度。 设置适当的值以获得显着的信号电平和足够的曲率。
        2. 在一些情况下,抗原从MAb非常缓慢地解离,然后我们需要设置非常长的洗涤期。 当没有观察到解离时,尝试降低MAb的固定水平。
      3. 抗原剥离条件。
      4. 空白实验设计:无MAb固定和/或注射HBS-EP缓冲液替代抗原溶液
      5. 单个流路的设置。
        注意:通常,SPR系统有多个流路用于空白实验和一次检查多个MAbs。

  4. 曲线分析
    1. 收集的实验数据根据SPR系统的供应商提供的拟合算法进行分析(例如,参见图1c)。通常使用1:1 Langmuir拟合模型 注意:检查软件提供的卡方值和质量指数。


      图1. SPR实验的实际示例 t 。
      1. 测定抗人Fcγ的胺偶联固定的pH值。 pH 4.0显示最高的反应,然而,它也显示出流平的迹象。然后,我们在这种情况下选择pH 4.5。打开的箭头和闭合的箭头分别表示注射抗人Fcγ溶液的开始和结束点。  
      2. 通过抗人Fcγ和人MAb之间的相互作用将MAb固定在传感器芯片上。 打开的箭头和闭合的箭头分别表示注射人MAb溶液的开始和结束点。 基线水平(在这种情况下,约10RU)的增量表示MAb的固定量
      3. 对于B型流感血凝素(HA)和人抗HA抗体之间的相互作用的代表性SPR反应。 打开的箭头表示HA溶液的注射,并对应于缔合期。 闭合箭头表示最终注射的结束,并对应于解离期的开始。 通过1:1 Langmuir拟合模型获得的拟合曲线也用灰线显示

笔记

  1. 通常,在集成系统中进行SPR分析,其中设备(传感器芯片,缓冲瓶,样品管等),实验设计,分析算法以及甚至试剂都供应用于使用该仪器。 我们在这里描述了抗原-MAb相互作用分析的SPR实验的一般程序。 请按照您的仪器手册和/或联系供应商以获得更详细的协议

食谱

  1. 0.5M HEPES缓冲液(pH7.4)(500ml) 将59.6g HEPES粉末与400ml dH 2 O混合 用NaOH调节pH至7.4 将dH <2> O添加到500ml
    用0.22μm底部过滤器和真空泵过滤
  2. 0.5M EDTA(pH8.0)(500ml) 将93.1g的EDTA缓冲液与400ml dH 2 O混合。
    用NaOH调节pH至8.0 将dH <2> O添加到500ml
    用0.22μm底部顶部过滤器过滤并在室温下贮存
  3. 20%(v/v)聚山梨醇酯20(500ml) 将400ml dH 2 O加入到100ml聚氧乙烯(20)脱水山梨糖醇单月桂酸酯中
  4. 10x HBS-E(500ml) 混合100ml 0.5M HEPES缓冲液,pH 7.4
    30ml 0.5M EDTA,pH8.0 44克NaCl
    将dH <2> O添加到500ml
  5. 1x HBS-EP(4 L)
    10x HBS-E缓冲液400ml
    将dH <2> O添加到4 L
    用0.22μm底部过滤器和真空泵过滤 加入1ml 20%(v/v)聚山梨醇酯20
  6. 400mM EDC(10ml) 将750mg EDC溶解在10ml dH 2 O中 分配200微升到1.5毫升管,并储存在-20°C
  7. 100mM NHS(10ml) 将115mg NHS溶解在10ml dH 2 O中 分配200微升到1.5毫升管,并储存在-20°C
  8. 1 M 2-乙醇胺(pH8.5)(50ml) 将3.0ml 2-乙醇胺用dH 2 O稀释至40ml
    用HCl
    调节pH至8.5 将dH <2> O添加到50ml
    分配1毫升到1.5毫升管和存储在4℃
  9. 50mM NaOH(10ml) 用dH 2 O至10ml稀释250μl2M NaOH溶液 将1ml分配到1.5ml管中并在室温下保存
  10. 10mM乙酸盐缓冲液(pH4.0,4.5,5.0,5.5)(各50ml) 将8.2g乙酸钠与100ml dH 2 O混合以制备1M乙酸盐/Na 用100ml dH 2 O稀释5.7ml乙酸盐以制备1M乙酸盐 用dH 2 O稀释100倍制备10mM乙酸钠和10mM乙酸钠。
    通过混合10mM乙酸盐和10mM乙酸盐-Na调节pH 用0.22μm过滤器和注射器过滤
  11. 甘氨酸缓冲液10mM(pH 1.8,2.0 2.2,2.4)(各50ml) 将0.30g甘氨酸与dH 2 O混合并将dH 2 O加至200ml
    将50ml甘氨酸溶液分配到100ml烧杯中
    加入HCl调节pH值
    将dH <2> O添加到100 ml
    用0.22μm过滤器和注射器过滤
  12. 1 M NaCl
    将2.9g NaCl与dH 2 O混合至50ml
    用0.22μm过滤器和注射器过滤
  13. 3 M MgCl 2
    用dH 2 O混合30.5g MgCl 2和6H 2 O,并将体积调节至50ml br/> 用0.22μm过滤器和注射器过滤

致谢

该方案通过参考Biacore T200(GE-Healthcare)和ProteOn XPR36(BIO-RAD)的产品手册建立。

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。
  • 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。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nishimura, M. and Ikuta, K. (2013). Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction. Bio-protocol 3(24): e1006. DOI: 10.21769/BioProtoc.1006.
  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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。