ToF-SIMS and PCA of surface-immobilized antibodies with different orientations

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.