title: Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay.
abstract: Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL-1for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
authors.forename: Hyo-Deok
authors.initials: HD
authors.lastname: Seo
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
authors.forename: Joong-Jae
authors.initials: JJ
authors.lastname: Lee
authors.affiliation: Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Yu Jung
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hantschel
authors.affiliation: Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea. Electronic address: hskim76@kaist.ac.kr.
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D000906:Antibodies / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
chemical_list: D000906:Antibodies; D007202:Indicators and Reagents; D014409:Tumor Necrosis Factor-alpha; D000469:Alkaline Phosphatase
keywords: Genetic fusion; Immuno-reagent; Immunoassay; Monomeric alkaline phosphatase; Repebody