title: Fluorophore-labeled nanocapsules displaying IgG Fc-binding domains for the simultaneous detection of multiple antigens.
abstract: Simultaneous detection of multiple antigens by conventional immunological methods has been limited by the source of primary antibodies. Each antibody should be derived from a different host species (or subclass of immunoglobulin (Ig)) for suppressing the cross-reactions of secondary antibodies. Here we describe an innovative method for simultaneous, rapid, and sensitive detection of multiple antigens using ∼30-nm bio-nanocapsules (BNCs) displaying IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). When Cy2-labeled ZZ-BNC (Cy2-ZZ-BNC) was used instead of Cy2-labeled secondary antibody in western blot analysis, both sensitivity and signal intensity were significantly increased. The complex of Cy5-ZZ-BNC and mouse IgG2a (which shows moderate affinity to the Z domain) was not dissociated, even in the presence of 8-fold excess of free mouse IgG2a. In addition, crosslinking with BS(3) (bis-sulfosuccinimidyl suberate) efficiently stabilized the interaction. The ZZ-BNCs labeled with various Cy dyes facilitated the simultaneous detection of multiple antigens using primary antibodies derived from the same host species, by western blot analysis, immunocytochemistry and flow cytometry, which could expand the possibility of bio-imaging probes in various immunofluorescence techniques.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Iijima
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsuzaki
authors.forename: Nobuo
authors.initials: N
authors.lastname: Yoshimoto
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Niimi
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis*; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D053769:Nanocapsules / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism
chemical_list: D000941:Antigens; D005456:Fluorescent Dyes; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D053769:Nanocapsules; D013205:Staphylococcal Protein A