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Sandwich ELISA is a highly sensitive method that can be used to determine if two epitopes are part of the same macromolecule or supramolecular complex. In the case of plant cell wall glycans, it can reveal the existence of inter-polymers linkages, leading to better understanding of overall cell wall architectures. This development of a conventional sandwich ELISA protocol uses a carbohydrate-binding module (CBM), a small protein domain found in some carbohydrate catalysing or activating enzymes, and rat monoclonal antibodies (mAbs) which can be combined in the same ELISA plate without risk of cross reaction; the secondary anti-rat HRP antibody being only able to bind to the rat mAb and not the CBM. This protocol was developed and modified in the Prof. J. Paul Knox lab at the University of Leeds.

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Sandwich Enzyme-linked Immunosorbent Assay (ELISA) Analysis of Plant Cell Wall Glycan Connections
夹心酶联免疫吸附实验(ELISA)分析植物细胞壁中的糖链连接

植物科学 > 植物生物化学 > 糖类
作者: Valérie Cornuault
Valérie CornuaultAffiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK
For correspondence: bsvrc@leeds.ac.uk
Bio-protocol author page: a1299
 and J. Paul Knox
J. Paul KnoxAffiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK
For correspondence: j.p.knox@leeds.ac.uk
Bio-protocol author page: a1300
Vol 4, Iss 8, 4/20/2014, 5754 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1106

[Abstract] Sandwich ELISA is a highly sensitive method that can be used to determine if two epitopes are part of the same macromolecule or supramolecular complex. In the case of plant cell wall glycans, it can reveal the existence of inter-polymers linkages, leading to better understanding of overall cell wall architectures. This development of a conventional sandwich ELISA protocol uses a carbohydrate-binding module (CBM), a small protein domain found in some carbohydrate catalysing or activating enzymes, and rat monoclonal antibodies (mAbs) which can be combined in the same ELISA plate without risk of cross reaction; the secondary anti-rat HRP antibody being only able to bind to the rat mAb and not the CBM. This protocol was developed and modified in the Prof. J. Paul Knox lab at the University of Leeds.

[Abstract] 夹心ELISA是高度灵敏的方法,其可以用于确定两个表位是否是相同大分子或超分子复合物的一部分。 在植物细胞壁聚糖的情况下,它可以揭示存在的聚合物之间的联系,导致更好地了解整体细胞壁结构。 常规夹心ELISA方案的这种发展使用碳水化合物结合模块(CBM),在一些碳水化合物催化或活化酶中发现的小蛋白结构域和大鼠单克隆抗体(mAb),其可以组合在相同的ELISA板中,没有风险 交叉反应; 次级抗大鼠HRP抗体仅能够结合大鼠mAb,而不能结合CBM。 该协议在利兹大学的J. Paul Knox实验室开发和修改。



Figure 1. Sandwich ELISA analysis of complex glycans

Materials and Reagents

  1. Cell wall extracts [50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383 ) or 4 M KOH/1% (w/v) NaBH4 (Sigma-Aldrich, catalog number: 452882 ) or purified cell wall polymers]
    Notes:
    1. Reagents for cell wall extracts are 50 mM cyclohexanediamine tetraacetic acid (CDTA) (Sigma-Aldrich, catalog number: D1383) or 4 M KOH/1% (w/v) NaBH4 (Sigma-Aldrich, catalog number: 452882)
    2. Cell wall extraction protocol using 4 M KOH/1% (w/v) NaBH4 is described in Cid et al. (2010). This extraction will disrupt and release most cell wall glycans.
    3. CDTA is a chelating agent which is used to specifically extract the pectin fraction from cell walls. The same protocol can be followed using 50 mM CDTA instead of 4 M KOH (no neutralizing step necessary).
  2. Skimmed milk powder (Marvel Original) [used at 5% (w/v) in 1x PBS]
  3. Purified recombinant carbohydrate-binding module (CBM)
    Note: A protocol for CBM expression and purification can be found in Lee et al. (2013).
  4. Primary monoclonal antibody (PlantProbes, www.plantprobes.net)
  5. Secondary antibody (depending on the origin of the primary antibody used)
    1. Anti-rat IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A9542 )
    2. Anti-mouse IgG-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich, catalog number: A6782 )
  6. 10 mg/ml tetramethyl benzidine (Sigma-Aldrich, catalog number: T2885 )
  7. 1 M sodium acetate buffer (pH 6)
  8. 6% H2O2 (VWR International, catalog number: 2858175C )
  9. 2.5 M H2SO4 (VWR International, catalog number: 191675A )
  10. Tween 20 (Sigma-Aldrich, catalog number: P2287 ) (optional)
  11. 1x phosphate-buffered saline (PBS) (pH 7) (see Recipes)
  12. HRP developing solution (see Recipes)

Equipment

  1. 96-well surface treated ELISA microtitre plates (Thermo Fisher Scientific, Nunc-Immnuno Maxisorp, catalog number: 442404 )
  2. Microtitre plate reader (450 nm absorbance) (Thermo Fisher Scientific, MultiskanTM FC Microplate Photometer)
  3. Multi-channel pipette (if available, not critical)
  4. pH meter

Procedure

  1. Coat the microtitre plate using 0.5 to 1 µg/ml CBM in 1x PBS, 100 µl per well at 4 °C overnight.
    Note: The amount of CBM necessary to obtain a good signal will vary depending of its affinity to the substrate. Trials may be necessary to find the appropriate dilution.
  2. Wash the plate 3 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
    Note: We routinely used tap water but one might prefer using 1x PBS 0.1 % (v/v) Tween 20. The drying step is critical as any remaining water/buffer would dilute the next reagent. Make sure the plates are fully dried after each washing step.
  3. Block the plate for 2 h using 5% (w/v) milk powder in 1x PBS, 200 µl per well at room temperature.
  4. Wash the plate 3 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
  5. Incubate for 90 min with a cell wall extract or test glycan diluted in 5% (w/v) milk/1x PBS, 100 µl per well at room temperature.
    Note: Normal ELISA analysis can be used to determine which dilution of the extract gives the best signal. However, the amount of CBM used will determine the amount of glycan that can be immobilised. Therefore it is better to use more concentrated sample rather than less.
  6. Wash the plate 6 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
  7. Incubate with a primary antibody appropriately diluted in 5% (w/v) milk/1x PBS, 100 µl per well for 1 h at room temperature.
    Note: Use 10x dilution of hybridoma cell culture supernatants if working with PlantProbes monoclonal antibodies.
  8. Wash the plate 6 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
  9. Incubate with secondary HRP-conjugated antibody diluted 1:1,000 in 5% (w/v) milk/1x PBS, 100 µl per well for 1 h at room temperature.
  10. Wash the plate 9 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
    Note: Plates should be thoroughly washed at this step to ensure no false positive signals due to non-specifically bound secondary antibody/HRP.
  11. Develop the plate using 150 µl of substrate solution per well to detect secondary antibody binding. Let the plates develop for 5 to 10 min. Stop the reaction by adding 50 µl of 2.5 M sulphuric acid.
    Note: As developing the plate is a time dependent process it is important to ensure that all comparable samples are developed for the same time.
  12. To collect the data, read the plates at 450 nm using the microtitre plate reader.

Notes

  1. Make sure all experimental combinations in the microtitre plate wells are assayed in triplicates.
  2. A control with no CBM (1x PBS) needs to be included in every experiment. This should remain blank if the plates are properly blocked. In case of a signal in no CBM wells increase the % of milk powder, block for longer or use >3% Bovine Serum Albumin in place of milk powder.

Recipes

  1. 1x phosphate-buffered saline (PBS) (pH 7)
    1. To prepare this buffer you need to add NaH2PO4 to 0.6712 moles/L and Na2HPO4 to 0.3288 moles/L.
    2. The amount of acid and base is calculated using the Henderson-Hasselbalch equation [pH= pKa + log (Base)/(Acid)].
    3. Prepare just under a litre of the solution using the correct amounts of NaH2PO4 and Na2HPO4 in dH2O.
    4. Check the pH using a pH meter and adjust slightly if necessary using phosphoric acid or sodium hydroxide.
    5. Once the pH is adjusted, bring the volume to 1 L.  
  2. HRP developing solution (50 ml)
    Mix 45 ml of dH2O with 5 ml of 1 M sodium acetate buffer (pH 6)
    Add 500 µl of tetramethyl benzidine
    At the last minute add 50 µl of 6% H2O2
    Note: Adding hydrogen peroxide starts the reaction. It is therefore important to add it at the last minute to avoid high background signal.

Acknowledgments

The research leading to this protocol has received funding from the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (Grant Agreement n 263916). This article reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein.

References

  1. Cid, M., Pedersen, H. L., Kaneko, S., Coutinho, P. M., Henrissat, B., Willats, W. G. and Boraston, A. B. (2010). Recognition of the helical structure of beta-1,4-galactan by a new family of carbohydrate-binding modules. J Biol Chem 285(46): 35999-36009.
  2. Lee, K. J., Cornuault, V., Manfield, I. W., Ralet, M. C. and Knox, J. P. (2013). Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls. Plant J 75(6): 1018-1027.



图1.复合糖蛋白的夹心ELISA分析

材料和试剂

  1. 细胞壁提取物[50mM环己二胺四乙酸(CDTA)(Sigma-Aldrich,目录号:D1383)或4M KOH/1%(w/v)NaBH 4 :452882)或纯化的细胞壁聚合物] 注意:
    1. 细胞壁提取物的试剂是50mM环己二胺四乙酸(CDTA)(Sigma-Aldrich,目录号:D1383)或4M KOH/1%(w/v)NaBH 4(Sigma-Aldrich,目录号:452882)
    2. 使用4M KOH/1%(w/v)NaBH 4的细胞壁提取方案描述于Cid et al。 (2010)。 这种提取将破坏和释放大多数细胞壁聚糖。
    3. CDTA是用于从细胞壁特异性提取果胶级分的螯合剂。 使用50mM CDTA代替4M KOH(不需要中和步骤)可以遵循相同的方案。
  2. 脱脂奶粉(Marvel Original)[以1%PBS中的5%(w/v)使用]
  3. 纯化的重组碳水化合物结合模块(CBM)
    注意:用于CBM表达和纯化的方案可以在Lee et al。 (2013)。
  4. 主要单克隆抗体(PlantProbes, www.plantprobes.net
  5. 二抗(取决于所用一抗的来源)
    1. 抗大鼠IgG-辣根过氧化物酶(HRP)缀合(Sigma-Aldrich,目录号:A9542)
    2. 抗小鼠IgG-辣根过氧化物酶(HRP)缀合(Sigma-Aldrich,目录号:A6782)
  6. 10mg/ml四甲基联苯胺(Sigma-Aldrich,目录号:T2885)
  7. 1M乙酸钠缓冲液(pH 6)
  8. 6%H 2 O 2(VWR International,目录号:2858175C)
  9. 2.5 M H sub SO SO 4(VWR International,目录号:191675A)
  10. 吐温20(Sigma-Aldrich,目录号:P2287)(任选)
  11. 1×磷酸盐缓冲盐水(PBS)(pH 7)(参见配方)
  12. HRP开发解决方案(参见配方)

设备

  1. 96孔表面处理的ELISA微量滴定板(Thermo Fisher Scientific,Nunc-Immnuno Maxisorp,目录号:442404)
  2. 微量滴定板读数器(450nm吸光度)(Thermo Fisher Scientific,Multiskan FC微孔板光度计)
  3. 多通道移液器(如果可用,不重要)
  4. pH计

程序

  1. 使用0.5至1μg/ml CBM的1×PBS,每孔100μl在4℃过夜包被微量滴定板。
    注意:获得良好信号所需的CBM的量将根据其对底物的亲和力而变化。 可能需要进行试验以找到适当的稀释度。
  2. 微量滴定板读数器(450nm吸光度)(Thermo Fisher Scientific,Multiskan FC微孔板光度计)
  3. 多通道移液器(如果可用,不重要)
  4. pH计

程序

  1. 使用0.5至1μg/ml CBM的1×PBS,每孔100μl在4℃过夜包被微量滴定板。
    注意:获得良好信号所需的CBM的量将根据其对底物的亲和力而变化。 可能需要进行试验以找到适当的稀释度。
  2. ...
  3. Incubate with a primary antibody appropriately diluted in 5% (w/v) milk/1x PBS, 100 µl per well for 1 h at room temperature.
    Note: Use 10x dilution of hybridoma cell culture supernatants if working with PlantProbes monoclonal antibodies.
  4. Wash the plate 6 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
  5. Incubate with secondary HRP-conjugated antibody diluted 1:1,000 in 5% (w/v) milk/1x PBS, 100 µl per well for 1 h at room temperature.
  6. Wash the plate 9 times using tap water or 1x PBS 0.1 % (v/v) Tween 20. Shake the plate dry.
    Note: Plates should be thoroughly washed at this step to ensure no false positive signals due to non-specifically bound secondary antibody/HRP.
  7. Develop the plate using 150 µl of substrate solution per well to detect secondary antibody binding. Let the plates develop for 5 to 10 min. Stop the reaction by adding 50 µl of 2.5 M sulphuric acid.
    Note: As developing the plate is a time dependent process it is important to ensure that all comparable samples are developed for the same time.
  8. To collect the data, read the plates at 450 nm using the microtitre plate reader.

Notes

  1. Make sure all experimental combinations in the microtitre plate wells are assayed in triplicates.
  2. A control with no CBM (1x PBS) needs to be included in every experiment. This should remain blank if the plates are properly blocked. In case of a signal in no CBM wells increase the % of milk powder, block for longer or use >3% Bovine Serum Albumin in place of milk powder.

Recipes

  1. 1×磷酸盐缓冲盐水(PBS)(pH 7)
    1. 为了制备该缓冲液,需要将NaH 2 PO 4 4加入到0.6712摩尔/L和Na 2 HPO 4中, 到0.3288摩尔/L
    2. 使用Henderson-Hasselbalch方程[pH = pKa + log(Base)/(Acid)]计算酸和碱的量。
    3. 使用正确量的NaH 2 PO 4和Na 2 HPO 4来制备刚好在1升溶液下, in dH 2 O。
    4. 使用pH计检查pH,如果需要,使用磷酸或氢氧化钠稍微调整。
    5. 一旦调整pH,将体积调至1L。
  2. HRP显影液(50ml)
    将45ml dH 2 O与5ml 1M乙酸钠缓冲液(pH 6)混合
    加入500μl四甲基联苯胺
    在最后一分钟,加入50μl的6%H 2 O 2 sub 2
    注意:加入过氧化氢可启动反应。 因此,在最后一分钟添加它以避免高背景信号是很重要的。

致谢

导致该议定书的研究得到了WallTraC项目(授予协议n 263916)下欧洲联盟第七框架计划(FP7 2007-2013)的资助。 本文仅反映作者的观点。 欧洲共同体对可能对本文所包含的信息进行任何使用不承担任何责任。

参考文献

  1. Cid,M.,Pedersen,H.L.,Kaneko,S.,Coutinho,P.M.,Henrissat,B.,Willats,W.G.and Boraston,A.B。(2010)。 通过新的碳水化合物结合家族识别β-1,4-半乳聚糖的螺旋结构 模块。 J Biol Chem 285(46):35999-36009。
  2. Lee,K.J.,Cornuault,V.,Manfield,I.W.,Ralet,M.C.and Knox,J.P。(2013)。 烟草种子胚乳细胞中果胶鼠李糖半乳糖醛酸I(RG-I)结构特征的多尺度空间异质性 墙壁。 植物J   75(6):1018-1027
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How to cite this protocol: Cornuault, V. and Knox, J. P. (2014). Sandwich Enzyme-linked Immunosorbent Assay (ELISA) Analysis of Plant Cell Wall Glycan Connections. Bio-protocol 4(8): e1106. DOI: 10.21769/BioProtoc.1106; Full Text



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