An ellipsometric study on the density and functionality of antibody layers immobilized by a randomly covalent method and a protein a-oriented method

Da Shin Wang, Chia Chen Chang, Shiue Ching Shih, Chii Wann Lin*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

9 Scopus citations

Abstract

Ellipsometry is used to quantitatively evaluate the antigen-sensing capability of immuno-surfaces. The ellipsometric measurement is a rapid, label-free, and in situ detection; however, quantification of analytes by ellipsometry is not unambiguous. The proper use of the effective thickness as a measure for the amount of analytes is addressed in this article. Taking the effective thickness as the quantification reference, we find that employing protein A to immobilize antibodies makes the antigen capture two to three times higher than the covalent immobilization technique using N-succinimidyl 4-maleimidobutyrate (GMBS). Besides, the results also suggest a uniform orientation of antibodies achieved by protein A. Increasing the density of surface antibodies would improve the sensitivity as well; however, the effect is more crucial for the protein A immuno-surface and appears to be less important for the GMBS immuno-surface. Therefore, the optimal immobilization strategy should include an orientation layer, such as protein A, and maximize the amount of oriented antibodies on the surface to further improve the efficiency for antigen detection.

Original languageEnglish
Pages (from-to)303-310
Number of pages8
JournalBiomedical Engineering - Applications, Basis and Communications
Volume21
Issue number5
DOIs
StatePublished - 10 2009
Externally publishedYes

Keywords

  • Antibody immobilization
  • Antibody orientation
  • Antigen capture
  • Ellipsometry
  • Protein A

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