搜索

Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR
表面等离子共振法测定原初杂交瘤样本中抗体与抗原的结合亲和力   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Blood
Jan 2013

Abstract

Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the resulting complex was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration (Pope et al., 2009). Then the interaction with the protein was monitored at several concentrations and its affinity towards the immobilized antibodies was determined with the corresponding KD obtained from classical kinetics analysis. This set-up avoids the avidity effects of the bivalent antibodies, allows the use of non-purified analytes with unknown concentrations and the specific capture of the antibodies in a similar stable covalent-orientated manner.

Materials and Reagents

  1. Dulbecco’s phosphate buffered saline (D-PBS) (Sigma-Aldrich, catalog number: D8537 ) as running buffer during immobilization and binding analysis
  2. Amine coupling kit (GE Healthcare, catalog number: BR-1000-50 ) containing:
    1. 750 mg N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
    2. 115 mg N-hydroxysuccinimide (NHS)
    3. 10.5 ml Ethanolamine-HCl (pH 8.5)
  3. Mouse antibody capture kit (GE Healthcare, catalog number: BR-1008-38 )
  4. 10 mM acetate buffer (pH 5) (GE Healthcare, catalog number: BR-1003-51 )
  5. 10 mM acetate buffer (pH 5.5) (GE Healthcare, catalog number: BR-1003-52 )
  6. 10 mM Glycine-HCl (pH 1.7) (GE Healthcare, catalog number: BR-1008-38) as regeneration buffer
  7. Reagents for immobilization (see Recipes)
  8. Ligands (see Recipes)
  9. Reagents for binding assay (see Recipes)
  10. Analyte (see Recipes)

Equipment

  1. Sensor chip with carboxyl groups available for the amine coupling reaction: Serie S sensor chip CM5 (GE Healthcare, catalog number: BR-1005-30 )
  2. Instrument for SPR analysis: Biacore T100 instrument and Evaluation Software (GE Healthcare)
  3. Micropipettes (100-1,000 µl) (Eppendorf, catalog number: 033023B )
  4. Micropipettes (10-100 µl) (Eppendorf, catalog number: 033021B
  5. Micropipettes (2-20 µl) (Eppendorf, catalog number: 033019B )
  6. Plastic vials (0.8 ml) (GE Healthcare, catalog number: BR-1002-12 )
  7. Rubber caps (type 3) (GE Healthcare, catalog number: BR-1005-02 )

Software

  1. Biacore T100 Evaluation Software (GE Healthcare)

Procedure

  1. Immobilization of the antibodies from hybridoma supernatants
    1. Prepare 167 µl of 30 µg/ml anti-mouse IgG solution in the 10 mM acetate buffer (pH 5).
    2. The capture reagent anti-mouse IgG then was immobilized in four flow cells (fc) using the amino coupling protocol and following the instructions described in the “Mouse antibody capture kit” using the PBS buffer as running buffer. At the end of the anti-mouse IgG immobilization, the regeneration solution was injected three times to remove any anti-mouse IgG non-covalent bound to the chip, leaving intact the covalent bound anti-mouse IgG.
      Note: Flow cells are formed when a microfluidic flow system is brought into contact with a sensor surface on which the ligand is immobilized. Here, the sample is injected separately through independent, unconnected flow cells, although other configurations as serial flow cells or hydrodynamic addressing flow cell systems are possible.
      The reagents and parameters used for the different steps (activation of the carboxymethyl groups of the chip surface, ligand immobilization, surface deactivation and regeneration) are compiled in Table 1.
      Note: An immobilization level of anti-mouse IgG around 9,000-10,000 Resonance Unit (RU) is usually obtained.

      Table 1. Immobilization procedure of the anti-mouse IgG antibody
      Cycle
      Reagent
      Flow cell (fc)
      Contact time (sec)
      Flow rate (µl/min)
      1
      1:1 (v/v) mixture of EDC (0.4 M) and NHS (0.1 M)
      1*, 2, 3, 4
      420
      5 µl/min
      Anti-mouse IgG
      1*, 2, 3, 4
      420
      5 µl/min
      1 M Ethanolamine-HCl (pH 8.5)
      1*, 2, 3, 4
      420
      5 µl/min
      2**
      10 mM Glycine-HCl (pH 1.7)
      1*, 2, 3, 4
      3 times 60
      50 µl/min
      Notes: *Flow cell 1 serves as a reference cell and is only immobilized with the anti-mouse IgG.
                **This step is done in a different cycle and it is not shown in the Figure 1 below.

    3. An example of the sensorgram obtained for the anti-mouse IgG immobilization is shown in Figure 1 below.


      Figure 1. Immobilization of the anti-mouse IgG on the sensor chip

    4. Prepare 300 µl of hybridoma supernatant solution 5-fold diluted by mixing 60 µl of the sample with 240 µl of 10 mM acetate buffer (pH 5.5).
    5. After the regeneration step, (in which the non-covalent bound anti-mouse IgG is removed from the surface without damaging non-covalent bound anti-mouse IgG), the antibodies from the hybridoma supernatants are captured non-covalently onto the surface via their Fc region by the anti-mouse IgG and the resulting complex is stabilized using a crosslink protocol independently on each flow cell.
    6. After antibody coupling, 3 successive injections of regeneration were done.
    7. The reagents and parameters used for cross-linking of the captured antibodies to the anti-mouse IgG (short activation of the carboxy groups, ligand immobilization, deactivation and regeneration) are compiled in Table 2.
      Note: A maximum binding level of the antibodies coupled to the anti-mouse IgG around 1,500-2,500 RU should be obtained. Multiple injections of the diluted hybridoma supernatants can be done for reaching this binding level.

      Table 2. Procedure of immobilization of the antibodies captured on the immobilized anti-mouse IgG antibody
      Cycle
      Reagent
      Flow cell (fc)
      Contact time (sec)
      Flow rate (µl/min)
      3
      1:1 (v/v) mixture of EDC (0.4 M) and NHS (0.1 M)
      2, 3 or 4
      30
      5 µl/min
      4
      Hybridoma supernatant antibody sample
      2, 3 or 4
      600-1,800
      5 µl/min
      5
      1 M ethanolamine-HCl (pH 8.5)
      2, 3 or 4
      420
      5 µl/min
      6
      10 mM Glycine-HCl (pH 1.7)
      2, 3 or 4 3 times
      60
      50 µl/min

    8. An example of the sensorgram obtained for the antibody sample coupling is shown in the Figure 2 below.
      Note: Alternatively, non-covalent capture of the antibodies without crosslinking can be performed by omitting the cycle 3 and cycle 5. For that, direct injection of the antibody sample is done after immobilization with the anti-mouse IgG, without performing an activation or deactivation of the surface. The non-covalent capture produces similar immobilization levels but the binding signal could decrease over cycles due to baseline drift during measurement and regeneration leading to lower dynamic range and decrease of the sensitivity of the assay.


      Figure 2. Coupling of antibody sample using the cross-linking protocol (cycle 4)

  2. Analyte preparation
    In order to have an accurate measurement, the analyte should be in the same buffer as the continuous flow buffer to minimize bulk refractive index differences and avoid the so called bulk effect, so that can lead to low signal-to-noise-ratios. This is often most easily achieved through dilution of a concentrated analyte stock into running buffer. Therefore, the protein was diluted in the running buffer at a concentration of 4,000, 2,000, 1,000, 5,00, 250, 125, 62.5, 31.25, 15.63 and 0 nM before analysis.

  3. Binding assay
    1. The binding assay was performed using PBS as running buffer.
    2. Each dilution of the analyte sample was injected over flow cells 1, 2, 3 and 4. Injections were performed starting from 0 (PBS buffer blank) and followed by the lowest to the highest concentration at a flow rate of 50 µl/min for 90 sec.
      Once the injection was finished, the dissociation was performed by passing running buffer over the chip during 600 sec.
      Note: The contact time of the association phase should be long enough to have a curvature shape on the sensorgram and the contact time of the dissociation phase should be long enough to have a flat sensorgram after dissociation phase.
    3. After association and dissociation of the analyte, a regeneration step was performed in order to remove the remaining bound analyte. The 10 mM Glycine-HCl (pH 1.7) was selected as the optimal regeneration solutions because it allows the removal of the analyte bound to the ligand without changing the activity of the immobilized ligand. This was confirmed by the equal responses obtained from the binding assays before and after regeneration. Regeneration was performed with one injection of 3 sec (2.5 µl) of the regeneration solution.
      An example of the binding assay is shown in Table 3 below.

      Table 3. Summary of the binding assay conditions
      Cycle
      Status
      Reagent
      Flow cell (fc)*
      Contact time (sec)
      Flow rate (µl/min)
      7
      Association
      Analyte (protein)
      (0 and 15.63-4,000 nM)
      2-1, 3-1 or 4-1
      90
      50

      Dissociation
      PBS buffer blank
      2-1, 3-1 or 4-1
      600
      50

      Regeneration
      10 mM Glycine-HCl (pH 1.7)
      2-1, 3-1 or 4-1
      3
      50
      Note: *Binding is studied on the sensorgrams subtracted with the reference flow cell fc1.

      A sensorgram of protein-antibody sample interaction is shown in Figure 3.


      Figure 3. Binding assay

  4. Data analysis
    Data analysis is performed with the Biacore T100 Evaluation Software.
    1. Binding to the reference flow cell 1 was subtracted from each sample curve and maximal response unit was measured at 90 sec post injection.
    2. Best fitting was then established for 5 concentration of protein using the software. In general a 1:1 model Langmuir was selected for the fitting based on the expected interaction; alternatively in some cases a two-state reaction model was chosen.
    3. The kinetics constants (on-rate ka and off-rate kd) as well as the affinity (in terms of equilibrium dissociation constant KD) were then calculated. An example of the final fitting results using 5 concentrations of protein and with a model 1:1 Langmuir fitting (black curves) is shown in the Figure 4 and Table 4.
      Note: The final affinity measurement reported for the protein- antibody sample interaction was selected based on the Chi-square (Chi2), U-value and quality index.
      The chi-square value is a quantitative measure of the closeness of fit, and in an ideal situation will approximate to the square of the short-term noise level. It is however difficult to recommend absolute values for acceptance limits for chi square: the values need to be considered from case to case, but it is advised to consider kinetics results with the lowest chi-square value.
      The U-value obtained using the 1:1 binding model fitting, is an additional indicator of the parameter significance. This is a parameter that represents the uniqueness of the calculated rate constants and Rmax, determined by testing the dependence of fitting on correlated variations between selected variables. Lower values indicate greater confidence in the results. A high value (above about 25) indicates that the reported kinetic constants contain no useful information. Finally, kinetic constants should be uniquely determined and within the limitation of the instrument.


      Figure 4. Affinity measurement

      Table 4. Example of data extracted from the fitting procedure using the Biacore T100 evaluation software


      Note: Rmax corresponds to the analyte binding capacity of the surface,
      tc corresponds to the flow rate independent part of the mass transfer constant,
      kt corresponds to the mass transport constant and is calculate as following:
      kt= km*MW*109
      Where km corresponds to the mass transport coefficient and is a function of the flow rate, flow cell dimensions and diffusion properties of the analyte,
      RI to the bulk refractive index contribution in the sample.

Notes

Please follow the manual of your SPR instrument to implement this protocol.
In our hands, the average ± standard deviation for immobilization levels from 31 binding assay analyses was 2,326 ± 558 RUs.

Recipes

The following recipes were used to prepare the polyclonal antibody samples from our hybridoma supernatants and protein that were optimal for our assay.

  1. Reagents for immobilization
    Running buffer, Dulbecco’s phosphate buffered saline buffer, modified without calcium chloride and magnesium chloride which contains:
    2.7 mM KCl
    1.5 mM KH2PO4
    136.9 mM NaCl
    8.9 mM Na2HPO4.7H2O
  2. Ligands
    Anti-mouse IgG (Polyclonal rabbit anti-mouse immunoglobulin) diluted in 10 mM acetate buffer (pH 5.0)
    Polyclonal antibody sample from hybridoma supernatants, diluted 5 fold in 10 mM acetate buffer (pH 5.5). The standard procedure for hybridome supernantant generation is the following: After the fusion, the motherclones are plated onto 96-well plates (typically 32 plates with 50,000 cells/well) and the fusions are kept in culture for 10-14 days until clones become visible and cells are ready for screening. Four days before screening, the cell medium is changed in order to avoid false-positive signals from antibodies produced by non-surviving clones. The supernatants from wells containing surviving hybridoma cells (i.e. the splenocytes which successfully fused with myeloma cells) are tested by Luminex (for the presence of antibodies with the desired binding profiles, likely “polyclonal” response) and selected motherclones are subcloned. Serial dilutions subcloning are done in an attempt to obtain single cell per well. Cells are left to grow for about two weeks and the wells containing single clones are tested for the presence of specific monoclonal antibodies. The stable, clonal hybridoma cells are expanded for 2 weeks in the serum-free cell culture medium. The supernatants are tested by Luminex to evaluate the concentration of the mouse IgG. Typically, the concentrations varied between 10 to 65 μg/ml. The example provided in Figure 4 and Table 4 is a polyclonal supernatant of a mother clone with a concentration of 20 μg/ml.
  3. Reagents for binding assay
    Running buffer, Dulbecco’s phosphate buffered saline buffer, modified without calcium chloride or magnesium chloride which contains:
    2.7 mM KCl
    1.5 mM KH2PO4
    136.9 mM NaCl
    8.9 mM Na2HPO4.7H2O
  4. Analyte
    Purified protein diluted in the running buffer at 0.5 mg/ml

References

  1. Canziani, G. A., Klakamp, S. and Myszka, D. G. (2004). Kinetic screening of antibodies from crude hybridoma samples using Biacore. Anal Biochem 325 (2): 301-307.
  2. Schraml, M. and Biehl, M. (2012). Kinetic screening in the antibody development process. Methods Mol Biol 901: 171-181.
  3. Pope, M. E., Soste, M. V., Eyford, B. A., Leigh Anderson, N., Pearson, T. W. (2009). Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. J Immunol Methods 341 (1-2): 86-96.

简介

表面等离子体共振(SPR)被广泛用于产生关于生物分子之间的特异性相互作用的动力学和亲和力信息。这种技术是无标签的,并实时监控绑定事件。它通常用于表征单克隆抗体 - 抗原相互作用。该方案特别描述了使用SPR与Biacore T100仪器测量粗杂交瘤样品对蛋白质的亲和力。为此,首先通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上(Canziani等人,2004; Schraml和Biehl,2012)。然后来自杂交瘤上清液的抗体通过其Fc区非共价地捕获到表面上,提供最佳的分析物结合方向。最后,通过与EDC/NHS交联来稳定所得的复合物,以避免在测量和再生期间的基线漂移(Pope等人,2009)。然后在若干浓度下监测与蛋白质的相互作用,并且用从经典动力学分析获得的相应K D确定其对固定化抗体的亲和力。这种设置避免了二价抗体的亲合力效应,允许使用具有未知浓度的非纯化分析物和以类似的稳定的共价定向方式特异性捕获抗体。

材料和试剂

  1. 在固定和结合分析过程中作为运行缓冲液的Dulbecco's磷酸盐缓冲盐水(D-PBS)(Sigma-Aldrich,目录号:D8537)
  2. 胺偶联试剂盒(GE Healthcare,目录号:BR-1000-50),其包含:
    1. 750mg N-乙基-N' - (3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)
    2. 115mg N-羟基琥珀酰亚胺(NHS)
    3. 10.5ml乙醇胺-HCl(pH8.5)
  3. 小鼠抗体捕获试剂盒(GE Healthcare,目录号:BR-1008-38)
  4. 10mM乙酸盐缓冲液(pH 5)(GE Healthcare,目录号:BR-1003-51)
  5. 10mM乙酸缓冲液(pH5.5)(GE Healthcare,目录号:BR-1003-52)
  6. 10mM甘氨酸-HCl(pH 1.7)(GE Healthcare,目录号:BR-1008-38)作为再生缓冲液
  7. 固定试剂(见配方)
  8. 配体(参见配方)
  9. 用于结合测定的试剂(参见配方)
  10. 分析物(参见配方)

设备

  1. 具有可用于胺偶联反应的羧基的传感器芯片:Serie S传感器芯片CM5(GE Healthcare,目录号:BR-1005-30)
  2. 用于SPR分析的仪器:Biacore T100仪器和评估软件(GE Healthcare)
  3. 微量移液管(100-1000μl)(Eppendorf,目录号:033023B)
  4. 微量移液管(10-100μl)(Eppendorf,目录号:033021B)
  5. 微量移液器(2-20μl)(Eppendorf,目录号:033019B)
  6. 塑料小瓶(0.8ml)(GE Healthcare,目录号:BR-1002-12)
  7. 橡胶帽(类型3)(GE Healthcare,目录号:BR-1005-02)

软件

  1. Biacore T100评估软件(GE Healthcare)

程序

  1. 来自杂交瘤上清液的抗体的固定化
    1. 准备167微升30微克/毫升抗小鼠IgG溶液在10毫米醋酸盐缓冲液(pH 5)
    2. 然后将捕获试剂抗小鼠IgG固定在四个流中 细胞(fc)使用氨基偶联方案和以下 使用PBS在"小鼠抗体捕获试剂盒"中描述的说明   缓冲区作为运行缓冲区。 在抗小鼠IgG的末端 固定,将再生溶液注射三次 去除与芯片非共价结合的任何抗小鼠IgG,保持完整  共价结合的抗小鼠IgG。
      注意:流动池形成时  使微流体流动系统与传感器接触 表面上固定有配体。这里,注入样品  分开通过独立的,未连接的流动池,虽然其他 配置为串联流动池或流体动力寻址流动池  系统是可能的。
      用于的试剂和参数 不同的步骤(激活芯片的羧甲基基团 表面,配体固定,表面失活和再生) 在表1中编译 注意:通常获得约9,000-10,000共振单位(RU)的抗小鼠IgG的固定水平。

      表1.抗小鼠IgG抗体的固定程序
      循环
      试剂
      流动池(fc)
      接触时间(秒)
      流速(μl/min)
      1
      EDC(0.4M)和NHS(0.1M)的1:1(v/v)混合物 1 *,2,3,4
      420
      5μl/min
      抗小鼠IgG
      1 *,2,3,4
      420
      5μl/min
      1M乙醇胺-HCl(pH8.5)
      1 *,2,3,4
      420
      5μl/min
      2 **
      10mM甘氨酸-HCl(pH 1.7)
      1 *,2,3,4
      3次60
      50μl/min
      注意:*流式细胞1作为参照细胞,只与抗小鼠IgG固定。
                **此步骤是以不同的周期完成的,如下图1所示。

    3. 用于抗小鼠IgG固定化获得的传感图的实例显示在下面的图1中。


      图1.抗小鼠IgG在传感器芯片上的固定

    4. 准备300微升的杂交瘤上清液5倍稀释 将60μl样品与240μl10mM乙酸盐缓冲液(pH 5.5)混合
    5. 在再生步骤后,(其中非共价键合 抗小鼠IgG从表面去除而不损害非共价的   结合的抗小鼠IgG),来自杂交瘤上清液的抗体 通过其Fc区非共价地捕获到表面上 抗小鼠IgG,并且使用交联稳定所得的复合物   协议独立地对每个流动池
    6. 抗体偶联后,连续进行3次再生
    7. 用于捕获的交联的试剂和参数 针对抗小鼠IgG的抗体(羧基的短激活) 基团,配体固定,失活和再生) 编译在表2中 注意:抗体的最大结合水平   应该获得约1500-2505RU的抗小鼠IgG偶联物。   可以进行稀释的杂交瘤上清液的多次注射 以达到该结合水平。

      表2.固定化在固定的抗小鼠IgG抗体上的抗体的固定程序
      循环
      试剂
      流动池(fc)
      接触时间(秒)
      流速(μl/min)
      3
      EDC(0.4M)和NHS(0.1M)的1:1(v/v)混合物 2,3或4
      30
      5μl/min
      4
      杂交瘤上清抗体样品
      2,3或4
      600-1,800
      5μl/min
      5
      1M乙醇胺-HCl(pH8.5)
      2,3或4
      420
      5μl/min
      6
      10mM甘氨酸-HCl(pH 1.7)
      2,3或4 3次
      60
      50μl/min

    8. 下面的图2显示了抗体样品偶联得到的传感图的一个例子 注意:   或者,非共价捕获抗体 可以通过省略循环3和循环5来进行交联 即,抗体样品的直接注射在之后进行 用抗小鼠IgG固定,而不进行活化   或表面的失活。 非共价捕获产生 类似的固定水平,但是结合信号可以减少   循环由于测量和再生期间的基线漂移 导致较低的动态范围和灵敏度的降低 测定。


      图2.使用交联方案(循环4)偶联抗体样品

  2. 分析物准备
    为了具有精确的测量,分析物应当在与连续流缓冲器相同的缓冲液中,以最小化体积折射率差异并避免所谓的体效应,从而可以导致低的信噪比。这通常最容易通过将浓缩的分析物原料稀释到运行缓冲液中来实现。因此,在分析前,蛋白质在运行缓冲液中以4,000,2,000,1,000,5,00,250,125,62.5,31.25,15.63和0nM的浓度稀释。

  3. 结合测定
    1. 使用PBS作为运行缓冲液进行结合测定
    2. 每 将分析物样品的稀释液注射到流动池1,2,3和4上 从0(PBS缓冲液空白)开始进行注射 随后以50的流速从最低到最高浓度  μl/min,90秒 一旦注射完成,通过使运行缓冲液在芯片上通过600秒进行解离。
      注意:   结合期的接触时间应足够长   传感图上的曲率形状和接触时间 解离相应足够长以具有平坦的传感图   解离相。
    3. 结合和解离后 分析物,进行再生步骤以除去 剩余结合分析物。 选择10mM甘氨酸-HCl(pH 1.7) 最佳的再生解决方案,因为它允许去除 分析物与配体结合而不改变其活性 固定配体。 这通过获得的相等响应来证实 从再生前后的结合测定。 再生是 进行一次注射3秒(2.5μl)的再生 解决方案 结合测定的实例显示于下表3中
      表3.结合测定条件的总结
      循环
      状态
      试剂
      流动池(fc)*
      接触时间(秒)
      流速(μl/min)
      7
      协会
      分析物(蛋白质)
      (0和15.63-4,000nM)
      2-1,3-1或4-1
      90
      50

      分裂
      PBS缓冲区空白
      2-1,3-1或4-1
      600
      50

      再生
      10mM甘氨酸-HCl(pH 1.7)
      2-1,3-1或4-1
      3
      50
      注意:*研究传感图与基准流动池fc1相减的结合。

      蛋白质 - 抗体样品相互作用的传感图如图3所示

      图3.结合测定


  4. 使用Biacore T100评估软件进行数据分析。
    1. 从每个样品中减去与参考流动池1的结合 在注射后90秒测量曲线和最大反应单位。
    2. 然后建立5个蛋白质浓度的最佳拟合 软件。 一般来说,选择1:1模型Langmuir 基于预期的相互作用拟合; 或者在某些情况下a   选择两状态反应模型
    3. 动力学常数 (on-rate k a 和off-rate )以及亲和力(根据 平衡解离常数K D)。 一个例子 的最终拟合结果使用5个浓度的蛋白质和a 模型1:1 Langmuir拟合(黑色曲线)如图4和图4所示 表4.
      注意:报告的最终关联性测量 蛋白质 - 抗体样品相互作用 卡方(Chi 2 ),U值和质量指数。
      卡方值 是一个定量测量的贴合度,并在一个理想的 情况将近似于短期噪声水平的平方。 然而,很难建议接受的绝对值 卡方的限制:需要考虑的值从案例到 情况,但建议考虑动力学结果最低 卡方值。
      使用1:1结合模型获得的U值 拟合,是参数意义的附加指标。这个 是表示计算的速率的唯一性的参数 常数和Rmax,通过测试拟合的依赖性来确定 相关变量。较低的值表示 对结果更有信心。高值(高于约25) 表明报道的动力学常数不含有用 信息。最后,动力学常数应该唯一确定 并在仪器的限制范围内。


      图4.亲和度测量

      表4.使用Biacore T100评估软件
      从拟合程序中提取的数据示例

      注意:R max 对应于表面的分析物结合能力 tc对应于质量传递常数的流速独立部分,
      对应于质量传递常数,计算如下: * MW * 10 9
      在哪里  k 对应于质量传递系数, 流量,流动池尺寸和扩散性能 分析物,
      RI对样品中的体积折射率贡献

笔记

请按照您的SPR仪器的手册来实施此协议。
在我们的手中,来自31个结合测定分析的固定水平的平均±标准偏差为2,326±558RU。

食谱

以下配方用于从我们的杂交瘤上清液和蛋白质制备多克隆抗体样品,其对于我们的测定是最佳的。

  1. 用于固定的试剂
    运行缓冲液,Dulbecco磷酸盐缓冲盐水缓冲液,不含氯化钙和氯化镁,其中含有:
    2.7 mM KCl
    1.5mM KH 2 PO 4 4/v/v 136.9mM NaCl 8.9mM Na 2 HPO 4 SubO 2·7H 2 O·h /
  2. 配体
    在10mM乙酸盐缓冲液(pH5.0)中稀释的抗小鼠IgG(多克隆兔抗小鼠免疫球蛋白) 来自杂交瘤上清液的多克隆抗体样品,在10mM乙酸盐缓冲液(pH 5.5)中稀释5倍。杂交体上清液产生的标准程序如下:融合后,将母克平板接种在96孔板(通常为具有50,000个细胞/孔的32个板)上,并将融合物保持在培养中10-14天直到克隆变得可见并且细胞准备用于筛选。在筛选前四天,更换细胞培养基以避免来自非存活克隆产生的抗体的假阳性信号。来自含有存活的杂交瘤细胞(即成功地与骨髓瘤细胞成功融合的脾细胞)的孔的上清液通过Luminex(用于存在具有所需结合谱的抗体,可能是"多克隆"应答)进行测试,并选择母克隆被亚克隆。进行连续稀释亚克隆以试图获得每个孔的单个细胞。使细胞生长约2周,并测试含有单个克隆的孔中是否存在特异性单克隆抗体。将稳定的克隆杂交瘤细胞在无血清细胞培养基中扩增2周。通过Luminex测试上清液以评价小鼠IgG的浓度。通常,浓度在10至65μg/ml之间变化。图4和表4中提供的实施例是浓度为20μg/ml的母克隆的多克隆上清液。
  3. 用于结合测定的试剂
    运行缓冲液,Dulbecco磷酸盐缓冲盐水缓冲液,不含氯化钙或氯化镁,其中含有:
    2.7 mM KCl
    1.5mM KH 2 PO 4 4/v/v 136.9mM NaCl 8.9mM Na 2 HPO 4 SubO 2·7H 2 O·h /
  4. 分析物
    在流动缓冲液中以0.5mg/ml稀释的纯化蛋白质

参考文献

  1. Canziani,G.A.,Klakamp,S。和Myszka,D.G。(2004)。 使用Biacore从粗杂交瘤样品中动力学筛选抗体。 Anal Biochem 325(2):301-307。
  2. Schraml,M。和Biehl,M。(2012)。 抗体开发过程中的动力学筛选 方法Mol Biol 901:171-181。
  3. Pope,M.E.,Soste,M.V.,Eyford,B.A.,Leigh Anderson,N.,Pearson,T.W。(2009)。 抗肽抗体筛选:通过精细表面等离振子共振技术选择高亲和力单克隆试剂。/a> J Immunol Methods 341(1-2):86-96。
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ndao, D. M., Hickman, D. T., López-Deber, M. P., Davranche, A., Pfeifer, A. and Muhs, A. (2014). Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR. Bio-protocol 4(21): e1276. DOI: 10.21769/BioProtoc.1276.
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,作者需要谷歌账户来上传视频。

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。