Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers

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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<sub>H</sub>) 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<sub>L</sub>) was added and incubated. The amount of immobilized SA-V<sub>L</sub> 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.

Journal

  • Analytical Sciences

    Analytical Sciences 29(9), 871-876, 2013-09-10

    The Japan Society for Analytical Chemistry

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Codes

  • NII Article ID (NAID)
    10031196097
  • NII NACSIS-CAT ID (NCID)
    AA10500785
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    09106340
  • NDL Article ID
    024830312
  • NDL Call No.
    Z54-F482
  • Data Source
    CJP  NDL  J-STAGE 
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