title: Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*.
abstract: The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Farhima
authors.initials: F
authors.lastname: Akter
authors.forename: Masayasu
authors.initials: M
authors.lastname: Mie
authors.forename: Sebastian
authors.initials: S
authors.lastname: Grimm
authors.forename: Per-Åke
authors.initials: PÅ
authors.lastname: Nygren
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000032:analysis* / Q000276:immunology; D001435:Bacteriophages / Q000737:chemistry; D015374:Biosensing Techniques; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay; D018796:Immunoconjugates / Q000737:chemistry; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013045:Species Specificity; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D018796:Immunoconjugates; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D007371:Interferon-gamma