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ToF‐SIMS and PCA of surface‐immobilized antibodies with different orientations
Author(s) -
Park JiWon,
Cho IlHoon,
Moon Dae Won,
Paek SeHwan,
Lee Tae Geol
Publication year - 2011
Publication title -
surface and interface analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.52
H-Index - 90
eISSN - 1096-9918
pISSN - 0142-2421
DOI - 10.1002/sia.3440
Subject(s) - biotinylation , chemistry , streptavidin , secondary ion mass spectrometry , analytical chemistry (journal) , chromatography , principal component analysis , antibody , biotin , mass spectrometry , biochemistry , biology , immunology , artificial intelligence , computer science
Abstract A number of studies have been done on the orientation of surface‐immobilized antibodies since it plays a significant role in the performance of immunoassays. Here, we present a new study by which the orientation of differently immobilized antibodies was directly probed by using time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and principal component analysis (PCA). For greater control over the orientations of intact IgG and F(ab′) 2 antibody fragment, they were either site‐directly biotinylated at the hinge region or randomly biotinylated at the amino groups, and were then immobilized onto a streptavidin‐terminated surface. According to the PCA results from ToF‐SIMS spectra, site‐directly and randomly biotinylated IgG became ‘end‐on’ oriented (Fc is closer to the surface) at a gradual rate as immobilization concentration increased, while site‐directly biotinylated IgG became oriented at a slightly faster rate. Furthermore, site‐directly biotinylated F(ab′) 2 quickly became ‘end‐on’ oriented even at a low concentration of 1 µg/ml as immobilization concentration increased, whereas randomly biotinylated F(ab′) 2 was not oriented at all over any concentration. Our results show that a ToF‐SIMS partnership with multivariate analysis is useful for interpreting protein orientations in terms of surface amino acid profiles. Copyright © 2010 John Wiley & Sons, Ltd.