title: Immuno-rolling circle amplification using a multibinding fusion protein.
abstract: Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.
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: Eiry
authors.initials: E
authors.lastname: Kobatake
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis*; D004270:DNA, Circular / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000032:analysis* / Q000276:immunology
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D014408:Biomarkers, Tumor; D004270:DNA, Circular; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; D001360:Avidin