title: Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A.
abstract: 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.
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Yumehiro
authors.initials: Y
authors.lastname: Hata
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Ninomiya
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ikura
authors.forename: Keigo
authors.initials: K
authors.lastname: Takeguchi
authors.forename: Satoka
authors.initials: S
authors.lastname: Aoyagi
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
mesh_terms: 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
chemical_list: D055910:Antibodies, Immobilized; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D012822:Silicon Dioxide