RRC ID 53882
Author Ohmuro-Matsuyama Y, Ueda H.
Title Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation.
Journal Anal Chem
Abstract The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigen-induced approximation of the two antibody variable region fragments VH and VL by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which VH and VL were each fused and expressed in Escherichia coli cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems.
Volume 90(5)
Pages 3001-3004
Published 2018-3-6
DOI 10.1021/acs.analchem.7b05140
PMID 29446920
MeSH Antibodies / immunology Base Sequence Immunoassay / methods* Immunoglobulin Variable Region / immunology Limit of Detection Luminescence Luminescent Measurements / methods* Osteocalcin / analysis Osteocalcin / immunology Peptides / analysis* Peptides / immunology
IF 6.785
Times Cited 3
DNA material pET-LnBiT (RDB16003)