Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A.
We previously reported a silica-binding protein, designated Si-tag, which can be used to immobilize proteins on silica surfaces. Here, we constructed a fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A for oriented immobilization of antibodies on a silicon wafer whose surface is oxidized to silicon dioxide (silica). The fusion protein, Si-tagged protein A, strongly bound to the silica surface with a dissociation constant of 0.31 nM. Time-of-flight secondary ion mass spectrometry analysis of the silicon wafer coated with Si-tagged protein A, combined with principal component analysis and mutual information, demonstrated that protein A is localized on the outermost surface of the bound protein layer. Immunoglobulin G (IgG) was immobilized both on the silicon wafer coated with Si-tagged protein A and, as a control, directly on the intact silicon wafer via physical adsorption. The silicon wafer coated with Si-tagged protein A bound 30-70% more IgG than the uncoated silicon wafer, whereas the antigen-binding activity was 4- to 5-fold higher for the former, indicating that IgG was functionally immobilized on the silicon wafer via Si-tagged protein A in an oriented manner.
Ikeda
Takeshi
T
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
Hata
Yumehiro
Y
Ninomiya
Ken-Ichi
K
Ikura
Yoshiaki
Y
Takeguchi
Keigo
K
Aoyagi
Satoka
S
Hirota
Ryuichi
R
Kuroda
Akio
A
D055910:Antibodies, Immobilized / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D007074:Immunoglobulin G / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D018629:Spectrometry, Mass, Secondary Ion; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry; D013499:Surface Properties
D055910:Antibodies, Immobilized; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D012822:Silicon Dioxide