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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

Mitsuhiro Nishimura Mitsuhiro Nishimura
Kazuyoshi Ikuta Kazuyoshi Ikuta

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1006 浏览次数: 13939

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2013

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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) search Monoclonal antibody (单克隆抗体) search IgG (IgG) search Viral antigen (病毒抗原) search Neutalization activity (neutalization活性) search

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

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 抗体分析 > 抗体-抗原相互作用

生物化学 > 蛋白质 > 相互作用 > 蛋白质-蛋白质相互作用

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