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
发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1106 浏览次数: 17483
评审: Renate Weizbauer
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参见作者原研究论文
The authors used this protocol in:
Sep 2013
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.
Figure 1. Sandwich ELISA analysis of complex glycans
Materials and Reagents
- 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:- 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)
- 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.
- 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).
- 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)
- Skimmed milk powder (Marvel Original) [used at 5% (w/v) in 1x PBS]
- Purified recombinant carbohydrate-binding module (CBM)
Note: A protocol for CBM expression and purification can be found in Lee et al. (2013). - Primary monoclonal antibody (PlantProbes, www.plantprobes.net)
- Secondary antibody (depending on the origin of the primary antibody used)
- 10 mg/ml tetramethyl benzidine (Sigma-Aldrich, catalog number: T2885 )
- 1 M sodium acetate buffer (pH 6)
- 6% H2O2 (VWR International, catalog number: 2858175C )
- 2.5 M H2SO4 (VWR International, catalog number: 191675A )
- Tween 20 (Sigma-Aldrich, catalog number: P2287 ) (optional)
- 1x phosphate-buffered saline (PBS) (pH 7) (see Recipes)
- HRP developing solution (see Recipes)
Equipment
- 96-well surface treated ELISA microtitre plates (Thermo Fisher Scientific, Nunc-Immnuno Maxisorp, catalog number: 442404 )
- Microtitre plate reader (450 nm absorbance) (Thermo Fisher Scientific, MultiskanTM FC Microplate Photometer)
- Multi-channel pipette (if available, not critical)
- pH meter
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
植物科学 > 植物生物化学 > 糖类
生物化学 > 糖类 > 多糖
生物化学 > 蛋白质 > 免疫检测
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