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Affinity Determination of Protein A Domains to IgG subclasses by Surface Plasmon Resonance
Linköping University, The Department of Physics, Chemistry and Biology.
2008 (English)Independent thesis Advanced level (degree of Magister), 20 points / 30 hpStudent thesis
Abstract [en]

A capture step with protein A is the most common purification step in the downstream purification process of monoclonal antibodies. It is therefore of great importance to increase the knowledge of the interactions involved in this purification technique. The purpose of this master thesis project was to determine the affinity of protein A domains to IgG subclasses by surface plasmon resonance (SPR).

Besides the five homologous IgG-binding protein A domains (E, D, A, B, and C) an engineered domain, similar to domain B and used in the protein A media MabSelect Sure™ (GE Healthcare) was included in the study. The domains were expressed in E.coli, affinity purified and immobilized onto sensor chip surfaces by amine coupling. The antibodies used in the interaction analyses were of the human IgG subclasses 1, 2, 3, and 4. Affinity determination was performed by kinetic analyses with the SPR-biosensor Biacore™ 2000.

All human IgG subclasses except IgG3 were shown to bind to all protein A domains including the monomer of the SuRe ligand. The equilibrium constants, KD-values, obtained were all in the low nanomolar range. For IgG1 and IgG4, no significantly differences in the affinity to any of the protein A domains were found, except for domain E where there might be quality issues of the prepared domain. Furthermore, a detected quality issue with the commercial IgG2 made it impossible to determine the KD-values for this subclass with any reliability.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2008. , 59 p.
Keyword [en]
Protein A, antibodies, IgG, SPR, Biacore, affinity determination
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:liu:diva-11340ISRN: LiTH-IFM-EX-08/1921-SEOAI: oai:DiVA.org:liu-11340DiVA: diva2:17753
Presentation
2008-02-22, Röntgen, Fysikhuset, Campus Valla, Linköping, 13:00
Uppsok
teknik
Supervisors
Examiners
Available from: 2008-03-25 Created: 2008-03-25

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CiteExportLink to record
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Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
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  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
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  • nn-NB
  • sv-SE
  • Other locale
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Output format
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