title: Protein-based open sandwich immuno-PCR for sensitive detection of small biomarkers.
abstract: Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-V(H)) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-V(L)) was added and incubated. The amount of immobilized SA-V(L) was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo.
authors.forename: Sharif
authors.initials: S
authors.lastname: Hasan
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.forename: Yuko
authors.initials: Y
authors.lastname: Hara
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Morizane
authors.forename: Futoshi
authors.initials: F
authors.lastname: Shibasaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis* / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D006801:Humans; D007118:Immunoassay; D015675:Osteocalcin / Q000032:analysis* / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000906:Antibodies; D000941:Antigens; D015415:Biomarkers; D015675:Osteocalcin