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A Chemiluminescence Based Receptor-ligand Binding Assay Using Peptide Ligands with an Acridinium Ester Label
使用吖啶酯标记的肽段配体进行化学发光的受体配体结合实验   

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Abstract

Studying the biochemical interaction of ligands with their corresponding receptors requires highly sensitive detection and monitoring of the bound ligand. Classically, radioactively labelled ligands have been widely used as highly sensitive tools for such binding measurements. Disadvantages of radiolabelling include instability of products, high costs and risks of working with radioactivity. Thus, assays using chemiluminescent probes offer convenient, highly sensitive alternatives. Here we suggest acridinium esters as suitable conjugates to label ligands of interest. Chemical oxidation of acridinium esters triggers chemiluminescence, allowing quantitation of this compound down to amol concentrations in standard luminometers. The first report about acridinium esters in immunoassays date back to 1983 (Weeks et al., 1983) and demonstrated the ability to conjugate acridinium to peptides, followed by using such peptides to measure receptor – peptide ligand interactions (Joss and Towbin, 1994).
Recently, this binding assay was adapted for studying derivatives of the plant peptide IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and their interaction with the corresponding receptor HSL2 (HAESA-LIKE 2) was reported (Butenko et al., 2014). Here we describe how this sensitive, nonradioactive binding approach can be used to reveal receptor-ligand binding in plant material.

Materials and Reagents

  1. Plant material harboring the receptor of interest in an immobilized form
    Suggested plant materials could be: Intact cells from suspension cultures, ground plant tissue or isolated receptors immobilized on immunoprecipitation beads.
    Notes:
    1. The receptor must be immobilized (on cells, cell debris, IP-beads) in order to allow efficient washing to remove unbound ligand without washing away the receptor molecules.
    2. Levels of some receptors are exceedingly low. Therefore, overexpression, e.g. by transient expression in Nicotiana benthamiana (N. benthamiana) leaves (Li, 2011), may be required to obtain sufficient binding sites.
    3. Use a negative control, same material lacking a functional receptor, whenever possible.
  2. MES (2-ethanesulfonic acid)
  3. NaCl
  4. DTT
  5. 33 µl proteinase Inhibitor (Sigma-Aldrich, catalog number: P9599 )
  6. 5 mM citric acid
  7. 0.03 % H2O2 in 100 mM NaOH (prepare fresh from a 30% H2O2 stock)
  8. Appropriate binding buffer (see Recipes)

Equipment

  1. Mortar and pestle
  2. Table top centrifuge
  3. Luminometer (e.g. single tube machines like FB 12, Berthold technologies, that allows injection)
  4. Acridinium-labeled peptide
    Important: The modified peptide should be tested for biological activity in your usually used bio-assay of choice and should not deviate much from the activity of the unmodified peptide.
  5. Unlabeled peptide (synthetic unlabeled peptides can be ordered by e.g. Biomatik)
  6. Peptides and conjugation with acridinium
    Note: Peptides can be ordered (on resin) from a company of choice or synthesized using solid-phase technology with Fmoc-protected amino acids. Acridinium esters can be conjugated to the N-terminal amino groups of the peptides on resin by coupling with N-hydroxysuccinimide activated acridinium esters (Cayman) before deprotection and purification of the peptides via HPLC.

Procedure

Note: Here, the procedure is described for using frozen, ground leaf tissue but the method should be easily adaptable for other materials.

  1. Shock-freeze leaf tissue (age does not matter, however, no senescence symptoms should be visible) or any material of choice (cell suspension culturue, e.g., N. benthamiana leaves in liquid nitrogen.
    Note: If using transient expression by agroinfiltration, or other transient expression methods, the time until your protein is expressed at high levels might vary, depending on the protein, promoter etc. Make sure to use a tag in order for expression level to be detected.
  2. Grind tissue in liquid N2 to a fine powder with mortar and pestle.
  3. Using a pre-cooled spatula, place the right amount of tissue in a pre-cooled 1.5 ml reaction tube. We use 500 mg tissue (frozen) for 10-12 individual samples/measurements (~ 40-50 mg per sample).
  4. Suspend in binding buffer (700-1,000 µl).
  5. Centrifuge suspension to create a pellet (13,000 x g, 1 min, 4 °C).
  6. Resuspend the pellet in binding buffer (500 mg tissue/ml binding buffer).
  7. Aliquot in 1.5 ml reaction tubes, 80 μl in each tube (n ≥ 3 per treatment). Cut off the tip of the pipette helps to avoid clogging of tissue at the tip and to get an even distribution of tissue in the samples.
  8. Add 20 μl of a solution containing the desired combination of acridinium labeled peptide and unlabeled peptide (peptides should be solved/diluted in binding buffer) to reach 100 μl.
    Note: Use a final concentration of 0.1 to 10 nM acridinium labeled peptide in your 100 µl sample. Run replicates (n ≥ 3) and samples containing an excess (>1,000-fold) of unlabeled peptide to determine non-specific binding (e.g. 1 nM Acridinium-peptide plus 1 µM unlabeled peptide as competitor).
  9. 20 min incubation (4 °C).
    Note: Reaching binding equilibrium/saturation may differ and experiments to optimize may be necessary. When no specific binding could be detected, parameters such as incubation time or temperature could be increased.
  10. Sediment the sample by centrifugation (13,000 x g, 1 min, 4 °C), pipette off and discard the supernatant.
  11. Wash the pellet 2-3 times by resuspension in 1 ml binding buffer and centrifugation (13,000 x g, 1 min, 4 °C).
    Note: Keep the total time required for these washing steps at a minimum! Depending on their dissociation rates, prolonged washing may lead to considerable loss of ligand binding.
  12. Resuspend the final pellet in 100 μl 5 mM citric acid.
  13. Place tube in the luminometer.
  14. Start the software and the program so it is ready to start collecting light immediately after induction with the NaOH/H2O2 solution.
    Note: Depending on your luminometer/software, you should monitor emitted light in a continuous mode or, if not possible in shortest possible intervals (e.g. each 0.2 sec or 0.5 sec) over time.
  15. Inject 100 μl of 100 mM NaOH containing 0.03% H2O2 (induction of luminescence) into the tube/cuvette containing your sample.
  16. Collect data for 10-30 sec.
    Note: The oxidation and light emission proceeds rapidly and should lead to a short flash of light (Joss and Towbin, 1994; Butenko et al., 2014). However, assay conditions and plant material may slow down the reaction. Nevertheless, integration time of 10-30 sec should be sufficient to collect > 90 % of the light emitted (Figure 1).

Representative data

Integrals of light emission can used to quantify the amount of ligand bound by comparison to an acridinium-ester standard curve.


Figure 1. Data set handling. Left panel: The collected data will give you a diagram showing the light (as RLU) collected over the time course of the experiments (left panel). By collecting the data points during the flash peak (shaded area in the figure) and integrating them, the data can be transferred to a diagram for comparison of the samples (to the right). Right panel: Obtained values (as described above) derived from different samples; control sample = plant material without receptor (e.g. from mutant plant, or untransformed plants) incubated with the same amount of Acridinium-labeled peptide and washed the same way as sample 1 and 2, respectively. Sample 1 and 2: grinded plant material with receptor was incubated with Acridinium-labeled peptide alone (black column) or with unlabeled peptide (1,000x excess) as competitor (grey column; + competitor). While the grey columns show the background of unspecific binding and the black column shows the total binding of the Acridinium-peptide the difference between the black and grey columns indicate the specific binding of Acridinium labeled peptide to the corresponding receptor within the respective sample(s).

Notes

  1. Additional notes and troubleshooting
    1. Standard curve with the particular acridinium-ligand and the photometer in use.
      1. Controls to test for light leakage and photoemission by plastic tubes etc should be performed.
      2. Kinetics of light emission should show a flash (~1 sec) and subsequent exponential decrease of light production.
      3. Integrals of emitted light (e.g. over 30 sec) should show (close to) linear relationship over several logs.
        → A sensitive photometer allows detection of a functional acridinium label down to the low fmol or amol range.
    2. Standard curves/values in the presence of the sample material and buffer conditions to be used in the actual binding assays.
      1. Controls to test for potential photoemission by the biological material (cells, tissue debris, conA beads, IP beads, etc.), buffers, detergents etc. in use should be added.
      2. Compare kinetics and integrals of light emission for standard concentrations to the values obtained in 1) to determine physical and chemical quenching.
        → Take quenching into account for determination of amounts of acridinium-labeled peptide present in the samples.
        Moderate quenching is inherent to these assays but strong quenching, e.g. > 80% light absorbed from leaf tissue or a strongly distorted kinetics of light emission require assay parameters to be re-adjusted. Such quenching could be avoided in a procedure that re-extracts the acridinium-label from the material before measuring.
    3. 'Non-specific' binding.
      1. Determine non-specific binding of the sample material (cells, tissue debris, conA beads, IP beads etc.) under the buffer-, timing-, washing- conditions to be used in the actual binding assays. Use either equivalent sample material containing no binding sites or add a high excess of non-labeled ligand (e.g. at concentrations ≥ 10 µM).
      2. Use these types of assays also to evaluate the washing steps (e.g. balance of pmoles of acridinium-labeled ligand added to the assays versus pmoles remaining after washing).
      3. The amount of non-specific binding depends on the amount of acridinium-labeled ligand applied (not necessarily in a linear way).
        → A certain amount of non-specific binding is inherent to all binding assays. Calculating the molar amounts that bind non-specifically indicate the minimal amounts of binding sites that have to be present in the samples in order to detect significant amounts of 'specific' binding.
        If necessary, minimize non-specific binding. This could be done by e.g. reducing the amount of acridinium-labeled ligand, changing sample material, ionic-conditions and buffering, or by addition of blocking substances to saturate unspecific binding sites on surfaces.
    4. 'Specific' binding = 'total' binding - 'non-specific' binding.
      Problems with detecting specific binding might be due to
      1. Amount of 'non-specific' binding sites > 'specific' binding sites.
      2. Low number of binding sites present (confirm presence of receptor molecules with immunological methods if possible).
      3. Low affinity of binding sites for the acridinium-labeled ligand could lead to loss of bound ligand during lengthy washing steps (washing time matters for reversible binding).
      4. Wrong/suboptimal conditions for binding [pH, ionic conditions, exposure of binding sites (e.g. vesicle formation), time, temperature].

  2. Final remarks
    The control parameters listed above define and limit the binding assays. Optimizing the experimental setup for studying the interaction between a given ligand and its particular receptor site might provide some repeated attempts. However, measuring of acridinium is fast and easy, therefore, once the setup is optimalized, several measurements and assay conditions can be tested per day.

Recipes

  1. Appropriate binding buffer
    Example of binding buffer used in (Butenko et al., 2014)
    25 mM MES (2-ethanesulfonic acid, pH 6.0)
    150 mM NaCl
    1 mM DTT (do not add DTT if receptors or ligands are sensitive to reducing agents)
    33 µl Proteinase Inhibitor mix per g plant material (essential)
    Note: The binding buffer might be adapted or modified by using different buffers adjusted to different pH (e.g. 25 mM MES pH 5.5 or 25 mM TRIS pH 7.5), different salt concentrations (reduce non-specific ionic interactions, e.g. 10, 50 or 100 mM NaCl) in order to optimize binding while keeping non-specific binding at a minimum.

Acknowledgments

R.A.’s work was supported by Grant 348256/F20 from the Research Council of Norway; and Grant 216856 from the Research Council of Norway and the Deutscher Akademischer Austausch Dienst. M. A. was supported by the Deutsch Forschungsgemeinschaft (AL1426/1-1). The method was recently described and applied in Butenko et al. (2014).

References

  1. Butenko, M. A., Wildhagen, M., Albert, M., Jehle, A., Kalbacher, H., Aalen, R. B. and Felix, G. (2014). Tools and strategies to match peptide-ligand receptor pairs. Plant Cell 26(5): 1838-1847.
  2. Joss, U. R. and Towbin, H. (1994). Acridinium ester labelled cytokines: receptor binding studies with human interleukin-1 alpha, interleukin-1 beta and interferon-gamma. J Biolumin Chemilumin 9(1): 21-28.
  3. Li, X. (2011). Infiltration of Nicotiana benthamiana protocol for transient expression via Agrobacterium. Bio-protocol Bio101: e95.
  4. Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K. and Woodhead, J. S. (1983). Acridinium esters as high-specific-activity labels in immunoassay. Clin Chem 29(8): 1474-1479.

简介

研究配体与其相应受体的生化相互作用需要对结合的配体的高度灵敏的检测和监测。通常,放射性标记的配体已广泛用作这种结合测量的高灵敏度工具。放射性标记的缺点包括产物的不稳定性,高成本和使用放射性的风险。因此,使用化学发光探针的测定提供方便,高度敏感的替代物。在这里,我们建议吖啶酯作为标记感兴趣的配体的合适的缀合物。吖啶酯的化学氧化触发化学发光,允许将该化合物定量至标准发光计中的amol浓度。关于免疫测定中的吖啶酯的第一次报道可以追溯到1983年(Weeks等人,1983),并且证明了将吖啶鎓结合到肽的能力,随后使用这样的肽测量受体 - 肽配体相互作用Joss和Towbin,1994)。最近,这种结合测定被改编用于研究植物肽IDA(消化缺陷缺陷)的衍生物,并且报道它们与相应的受体HSL2(HAESA-LIKE 2)的相互作用(Butenko等人,/em>,2014)。在这里我们描述如何这种敏感,非放射性绑定办法可用于揭示植物材料中的受体配体结合。

材料和试剂

  1. 含有固定化形式的感兴趣受体的植物材料
    建议的植物材料可以是:来自悬浮培养物的完整细胞,地面植物组织或固定在免疫沉淀珠上的分离的受体。 注意:
    1. 受体必须固定在(在细胞,细胞碎片,IP珠上) 以允许有效洗涤以除去未结合的配体 洗掉受体分子。
    2. 一些受体的水平 是非常低的。 因此,过表达,例如。 通过瞬态 在烟草(本塞姆氏烟草)叶中的表达(Li,2011), 可能需要获得足够的结合位点。
    3. 尽可能使用阴性对照,同样的材料缺乏功能受体。
  2. MES(2-乙磺酸)
  3. NaCl
  4. DTT
  5. 33μl蛋白酶抑制剂(Sigma-Aldrich,目录号:P9599)
  6. 5mM柠檬酸
  7. 0.03%H 2 O 2在100mM NaOH中的溶液(从30%H 2 O 2储备液中新鲜制备) )
  8. 适当的结合缓冲液(参见配方)

设备

  1. 砂浆和杵
  2. 台式离心机
  3. 发光计(例如单管机器,如FB 12,Berthold技术,允许注入)
  4. 吖啶标记肽
    重要:修饰的肽应在通常使用的生物测定中测试其生物活性,并且不应与未修饰肽的活性有太大的差异。
  5. 未标记的肽(合成的未标记肽可以通过例如 [Biomatik]订购)
  6. 肽和与吖啶鎓的共轭
    注意:肽可以从所选公司订购(在树脂上),或者使用固相技术用Fmoc保护的氨基酸合成。 吖啶酯可以在通过HPLC脱保护和纯化肽之前通过与N-羟基琥珀酰亚胺活化的吖啶酯(Cayman)偶联,在树脂上与肽的N-末端氨基缀合。

程序

注意:这里描述了使用冷冻的地面叶组织的方法,但是该方法应该容易适用于其他材料。

  1. 冲击冷冻叶组织(年龄无关紧要,但是,没有衰老症状应当可见)或任何选择的材料(细胞悬浮培养物,例如,本生烟草叶在液氮中 注意:如果通过农杆菌浸润或其他瞬时表达方法使用瞬时表达,直到蛋白质以高水平表达的时间可能不同,这取决于蛋白质,启动子等。确保使用标签以便表达水平被检测。
  2. 用研钵和研杵将液体N 2中的组织研磨成细粉末
  3. 使用预冷刮刀,将适量的组织放入预冷却的1.5ml反应管中。我们使用500毫克组织(冷冻)10-12个单独的样品/测量(〜40-50毫克每个样品)
  4. 悬浮在结合缓冲液(700-1,000微升)。
  5. 离心悬浮液以产生沉淀(13,000×g,1分钟,4℃)。
  6. 在结合缓冲液(500mg组织/ml结合缓冲液)中重悬沉淀
  7. 在1.5ml反应管中等分,每管80μl(每次处理n≥3)。切除移液管的尖端有助于避免尖端处的组织堵塞,并且使样品中的组织均匀分布。
  8. 加入20微升含有吖啶标记的肽和未标记的肽的所需组合的溶液(肽应该溶解/稀释在结合缓冲液中)以达到100μl。
    注意:在100μl样品中使用终浓度为0.1到10 nM吖啶标记的肽。运行重复(n≥3)和含有过量(> 1000倍)未标记肽的样品以确定非特异性结合(例如1nM吖啶 - 肽加上1μM未标记的肽作为竞争剂)。
  9. 20分钟孵育(4℃) 注意:达到结合平衡/饱和度可能不同,并且可能需要进行优化的实验。当没有检测到特异性结合时,可以增加参数如孵育时间或温度。
  10. 通过离心沉淀样品(13,000×g,1分钟,4℃),移液并弃去上清液。
  11. 通过重悬于1ml结合缓冲液中并离心(13,000×g,1分钟,4℃)来洗涤沉淀2-3次。
    注意:将这些洗涤步骤所需的总时间保持在最低限度!根据其解离速率,延长的洗涤可能导致配体结合的相当大的损失。
  12. 将最终沉淀重悬在100μl5mM柠檬酸中
  13. 将管放置在光度计中。
  14. 启动软件和程序,使其准备好在用NaOH/H 2 O 2溶液诱导后立即开始收集光。
    注意:根据您的光度计/软件,您应该以连续模式监测发射的光,或者如果不可能以最短的时间间隔(例如每0.2秒或0.5秒)监视发射光。
  15. 将含有0.03%H 2 O 2(诱导发光)的100μl的100mM NaOH注入到含有样品的试管/比色杯中。
  16. 收集数据10-30秒。
    注意:氧化和光发射快速进行,并应导致短暂的闪光(Joss和Towbin,1994; Butenko等,2014)。然而,测定条件和植物材料可以减慢反应。然而,10-30秒的积分时间应该足以收集> 90%的光发射(图1)。

代表数据

光发射的积分可以用于通过与吖啶鎓 - 酯标准曲线比较来定量结合的配体的量。


图1.数据集处理。左面板:收集的数据将显示在实验时间过程中收集的光(以RLU为单位)(左图)。通过在闪光峰值期间收集数据点(图中的阴影区域)并对其进行积分,可以将数据传输到用于比较样品的图(向右)。右图:从不同样品得到的获得值(如上所述);对照样品=用相同量的吖啶标记的肽孵育的没有受体(例如来自突变植物或未转化的植物)的植物材料,并分别以与样品1和2相同的方式洗涤。样品1和2:将带有受体的研磨植物材料与单独的吖啶标记的肽(黑色柱)或与未标记的肽(1,000x过量)作为竞争剂(灰色柱; +竞争剂)一起温育。虽然灰色柱显示非特异性结合的背景,黑色柱显示吖啶鎓肽的总结合,黑色和灰色柱之间的差异表明吖啶鎓标记的肽与相应样品内的相应受体的特异性结合, 。

笔记

  1. 其他说明和疑难解答
    1. 使用特定吖啶鎓配体和光度计的标准曲线
      1. 应该进行用于测试塑料管等的漏光和光发射的控制
      2. 光发射的动力学应该显示闪光(〜1秒)和随后的光产生的指数减少
      3. 发射光的积分(例如超过30秒)应该在几个日志上显示(接近)线性关系。
        →灵敏的光度计允许检测功能性吖啶标记低至fmol或amol范围。
    2. 在实际结合测定中使用的样品材料和缓冲液条件下的标准曲线/值
      1. 控制以测试生物的潜在光电发射 材料(细胞,组织碎片,conA珠,IP珠等),缓冲液, 洗涤剂等。 。
      2. 比较动力学和 标准浓度的光发射积分值 在1)中获得,以确定物理和化学淬火 →考虑猝灭以确定样品中存在的吖啶鎓标记的肽的量 中等猝灭是这些测定法所固有的,但是强烈的猝灭。 > 80%的光从叶组织吸收或强烈扭曲 光发射的动力学需要重新调整的测定参数。 这种淬灭可以在重新提取的过程中避免 吖啶标记从测量前的材料。
    3. "非特异性"结合。
      1. 确定样品材料(细胞,组织)的非特异性结合   碎片,conA珠,IP珠等)在缓冲液,定时,洗涤,   条件用于实际结合测定中。 使用 不含结合位点或添加高的等效样品材料 过量的未标记配体(例如在浓度≥10μM时)。
      2. 使用这些类型的测定法还评价洗涤步骤(例如,添加到测定中的吖啶鎓标记的配体的pmoles的平衡 与洗涤后剩余的pmol相比)。
      3. 大量的 非特异性结合取决于吖啶标记的配体的量 应用(不一定以线性方式)。
        →一定量 非特异性结合是所有结合测定所固有的。 计算 结合的摩尔量非特异性地指示最小量   结合位点,其必须存在于样品中以便检测   大量的"特定"约束力 如有必要,尽量减少 非特异性结合。 这可以通过例如减少金额来完成 吖啶标记的配体,改变样品材料,离子条件 和缓冲,或通过加入阻断物质饱和 表面上的非特异性结合位点。
    4. 'Specific'binding ='total'binding - 'non-specific'binding。
      检测特定绑定的问题可能是由于
      1. "非特异性"结合位点的量> '特异性'结合位点
      2. 存在低结合位点数(如果可能,用免疫学方法证实受体分子的存在)
      3. 结合位点对吖啶鎓标记的配体的低亲和力 可在长时间洗涤步骤(洗涤)期间导致结合配体的损失   时间问题,可逆绑定)。
      4. 错误/次优 结合条件[pH,离子条件,结合位点的暴露 (例如囊泡形成),时间,温度]。

  2. 最后评论
    上面列出的控制参数定义和限制结合测定。 优化用于研究给定配体与其特定受体位点之间的相互作用的实验装置可能提供一些重复的尝试。 然而,吖啶鎓的测量是快速和容易的,因此,一旦设置被最佳化,可以每天测试几种测量和测定条件。

食谱

  1. 适当的结合缓冲液
    (Butenko等人,2014)中使用的结合缓冲液的实例
    25mM MES(2-乙磺酸,pH 6.0) 150mM NaCl 1 mM DTT(如果受体或配体对还原剂敏感,则不添加DTT)
    33微升蛋白酶抑制剂混合物/g植物材料(必需)
    注意:结合缓冲液可以通过使用调节到不同pH的不同缓冲液(例如25mM MES pH 5.5或25mM TRIS pH 7.5),不同盐浓度(减少非特异性离子相互作用,例如10, 50或100mM NaCl)以优化结合,同时将非特异性结合保持在最小。

致谢

挪威研究委员会的Grant 348256/F20支持R.A.的工作;和来自挪威研究委员会和Deutscher Akademischer Austausch Dienst的Grant 216856。 M. A.由Deutsch Forschungsgemeinschaft(AL1426/1-1)支持。该方法最近被描述并应用于Butenko等人。 (2014年)。

参考文献

  1. Butenko,M.A.,Wildhagen,M.,Albert,M.,Jehle,A.,Kalbacher,H.,Aalen,R.B.and Felix,G。(2014)。 匹配肽配体的工具和策略 受体对。植物细胞 26(5):1838-1847。
  2. Joss,U.R.和Towbin,H。(1994)。 吖啶酯标记的细胞因子:与人白细胞介素-1α,白细胞介素-1β和干扰素的受体结合研究 γ-生物素化学发光物9(1):21-28。
  3. Li,X。(2011)。 通过土壤杆菌介导烟草的 方案的瞬时表达。 生物协议 Bio101:e95。
  4. Weeks,I.,Beheshti,I.,McCapra,F.,Campbell,A.K.and Woodhead,J.S。(1983)。 吖啶酯作为免疫测定中的高特异性活性标记。 Clin Chem 29(8):1474-1479
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引用:Wildhagen, M., Butenko, M. A., Aalen, R. B., Felix, G. and Albert, M. (2015). A Chemiluminescence Based Receptor-ligand Binding Assay Using Peptide Ligands with an Acridinium Ester Label. Bio-protocol 5(6): e1422. DOI: 10.21769/BioProtoc.1422.
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